LTB4 Promotes Insulin Resistance in Obese Mice by Acting On

© 2015 Nature America, Inc. All rights reserved. Beijing, Beijing, China. Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 obesity in state inflammatory tissue to the contribute also immune cell such types, as and eosinophils, lymphocytes, neutrophils variety of factors that could impair insulin signaling. In a addition, secrete other and number cell total the of 40% for account can rophages resistance insulin induced this chronic state tissue inflammatory has a key causal role in obesity- tissue adipose tissues target insulin the example, in obesity, there is a greater accumulation of immune cells in dence supporting this concept has gradually increased since then ago years of number a proposed was resistance insulin of to insulin resistance and aling, such other mechanisms, as lipotoxicity, can contribute also noids, and other factors in participate causing insulin sign decreased eicosa mediators such as cytokines, Inflammatory tissues. metabolic pathophysiologic defect characterized by impaired insulin signaling in complex a is resistance Insulin epidemic. diabetes 2 type parallel the driving is countries westernized in epidemic obesity current the and 2 mellitus type diabetes with patients of feature characteristic a is resistance Insulin leads to robust effects. insulin-sensitizing the LTB4–Ltb4r1 signaling pathway in hepatocyte and myocyte insulin resistance, and they show that were dependent on G accompanied by lower Akt insulin-stimulated and phosphorylation higher Irs-1/2 serine and phosphorylation, all of these events L6 myocytes, and impaired suppression of insulin-mediated hepatic glucose output in primary mouse hepatocytes. This was directly enhanced macrophage chemotaxis, stimulated inflammatory pathways, reduced glucose insulin-stimulated uptake in to an phenotype with anti-inflammatory protection from insulin resistance and hepatic steatosis. fat diet (HFD)–fed mice. Inhibition of the LTB4 receptor Ltb4r1, through either genetic or loss pharmacologic of function, led signaling. We found that liver, muscle and adipose tissue exhibit higher levels of the chemotactic eicosanoid LTB4 in obese high- Insulin resistance results from several mechanisms, including pathophysiologic chronic tissue inflammation and defective insulin Sayaka Taguchi Osborn Olivia Pingping Li on macrophages, hepatocytes and myocytes LTB4 promotes insulin resistance in mice obese by acting nature medicine nature Received 12 August 2014; accepted 13 January 2015; published online 23 February 2015; receptor termed Ltb4r1 or Ltb4r2 (refs. (refs. Ltb4r2 or protein–coupled Ltb4r1 termed G receptor a to binding by cytokines proinflammatory of and regulation the chemotaxis of promotion leukocyte the including A of leukotriene and protein, activities 5-lipoxygenase–activating sequential 5-lipoxygenase, the through acid arachidonic from erated Division Division of & Endocrinology Metabolism, Department of Medicine, University of California, San Diego, California, USA. 4 Leukotriene B Leukotriene hydrolase 3 1 These These authors contributed equally to this work. should Correspondence be addressed to P.L. ( 8 1 . LTB4 exerts well-characterized biological actions, actions, biological well-characterized exerts LTB4 . – 3 4 1– (LTB4) is a proinflammatory lipid mediator gen mediator lipid proinflammatory a is (LTB4) 1 , , Da Young Oh

4 , , Andrew Johnson 1 5 . Obesity is the major cause of insulin resistance, resistance, insulin of cause major the is Obesity . , , , Min Lu 6 advance online publication online advance . A role for proinflammatory signaling as a cause a i i and Jnk1, two downstream mediators of Ltb4r1 signaling. These observations elucidate a novel role of 1 4 . In visceral . fat mac In from subjects, visceral obese 1 & Jerrold M Olefsky 1 , 3 10 , , Gautam Bandyopadhyay 19– , 1 1 1 , , Heekyung Chung , , liver 2 1 ). Ltb4r1 (also known known (also Ltb4r1 ). 1 2 and muscle

9 7 1 , , 15– 8 and evi and 1 1 7 3 9 . , , and . For 1 , Rafael , MayoralRafael - - - - -

1 centrations of LTB4 (2–3 fold) in muscle, liver, and adipose tissue, tissue, adipose and liver, muscle, in fold) (2–3 LTB4 of con centrations higher exhibit mice obese HFD-induced that found have We Inhibition of Ltb4r1 improves metabolism RESULTS resistance. insulin and inflammation of treatment the for target drug valuable a be could Ltb4r1 that suggest findings these of All hepatocytes. and myocytes in signaling insulin decreased induce LTB4 directly can actions, chemotactic and proinflammatory potent Moreover, mouse models. found that, apart from its we unexpectedly small- obese genetic-mediated and diet-induced in both selective resistance, insulin Ltb4r1 dysfunction. an that metabolic inhibitor report molecule in we study, LTB4 current for the role In a indicating resistance insulin thereby and inflammation from protected were mice arthritis and atherosclerosis including pathologies, of inflammatory acteristic char inflammation persistent to contribute also can system Ltb4r1 of the LTB4–activation Chronic infection. acute during host defense is ubiquitously more and expressed receptor low-affinity a is (Blt2) Ltb4r2 osteoclasts. and cells T cells (T 1 helper type differentiated is macrophages, eosinophils, it granulocytes, and LTB4, for including cells, immune and specific inflammatory of variety a in receptor expressed high-affinity the is Blt1) as , , William S Lagakos It has reported that the LTB4–Ltb4r1 axis has an important role in role an has important axis LTB4–Ltb4r1 the that It reported has doi:10.1038/nm.380 22– H 1 1 cells), T 2 4 . One recent study showed that that showed study recent One . 1 , , Michael Maris 26– H [email protected] 2 2 and T 3 0 markedly reduces tissue inflammation and and inflammation tissue reduces markedly 0 1 , , Saswata Talukdar 19– H 2 Present Present address: State Key Laboratory of 17 17 cells, CD8 effector T cells, dendritic 2 1 In In vitro . ) ) or J.M.O. ( 1 , , Jachelle M Ofrecio in in vivo treatment with LTB4 [email protected] Ltb4r1 inhibition of Ltb4r1 1 s e l c i t r a ,

-knockout (KO) (KO) -knockout ). 1 , 2 5 - - ,

© 2015 Nature America, Inc. All rights reserved. Bonferroni Bonferroni in levels (Ins) insulin ( inhibitor. Ltb4r1 or vehicle with treated mice HFD-fed in studies glucose-clamp the in suppression ( inhibitor. Ltb4r1 ( inhibitor. Ltb4r1 ( inhibitor. Ltb4r1 or vehicle with Ltb4r1 ( mice. 1 Figure genetic deletion of to WT mice ( glucose-lowering effects of in insulin were better ( mice greater WT the to markedly incompared KOmice thethe were KO mice compared hyperinsulinemia and tolerance glucose ( obesity of degree same the achieved mice (WT) on a HFD (60% kcal from fat) for 12 weeks, obesity- in placed When system resistance. insulin and LTB4–Ltb4r1 inflammation tissue the associated of role the assess to studies loss-of-function pharmacologic-based and genetic- conducted we compared to chow-fed lean mice ( A WT. versus KO or inhibitor versus c a

f i –1

FFA suppression (%) s e l c i t r Body weight (g) Ins (ng ml ) LTB4 20 40 60 80 –1 10 15 20 25 20 40 60 0 (pmol 100 mg ) 0 0 5 0.05 0.10 0.15 0.20 0.25 -KO mice. ( mice. -KO b ) Body weight and glucose tolerance test of WT and and WT of test tolerance glucose and weight ) Body

Vehicle 0 Inhibition of Ltb4r1 leads to an insulin-sensitive phenotype. ( phenotype. insulin-sensitive an to leads Ltb4r1 of Inhibition Vehicle post hoc post WT Fig. Fig. 1 NC Week Week Inhibitor Muscl h g d Inhibito ) Glucose infusion rate (GIR) and insulin stimulated-glucose disposal rate (IS-GDR) of HFD-fed WT mice treated with vehicle or vehicle with treated mice WT HFD-fed of (IS-GDR) rate disposal stimulated-glucose insulin and (GIR) rate infusion ) Glucose ) Hepatic glucose production (HGP) suppression and HGP of HFD-fed WT mice treated with vehicle or Ltb4r1 inhibitor. ( inhibitor. Ltb4r1 or vehicle with treated mice WT HFD-fed of HGP and suppression (HGP) production glucose ) Hepatic Ltb4r1 KO tests. Values are expressed as mean mean as expressed are Values tests. ) Glucose tolerance test of HFD-fed mice treated with vehicle or Ltb4r1 inhibitor. ( inhibitor. Ltb4r1 or vehicle with treated mice HFD-fed of test tolerance ) Glucose ** c 2 0 * ), ), indicating an phenotype upon insulin-sensitive ob/ob e HF r

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1 0 * 120 HFD regimen, the mice were given vehicle or the Ltb4r1 inhibitor (50 inhibitor mg kg Ltb4r1 the or vehicle given were mice the regimen, HFD the of 12 week at starting and, weeks 14 for HFD mice WT Wefed Ltb4r2 (ref. (ref. Ltb4r2 CP105696 inhibitor (refs. Ltb4r1-specific the with mice WT treated HFD-fed we male medicine, translational successful for potential its study previous a with consistent 1 Fig. ( mice chow-fed lean in metabolism glucose on KO HF To further define the role of the LTB4–Ltb4r1 system and to role the of Tosystem assess LTB4–Ltb4r1 the define further Inhibito a ** D , 3 5 f ** Inhibitor Vehicle ** – 26– i ). These results in the HFD-fed HFD-fed the in results These ). ); ); Time (min) r c b n * ) Serum insulin level and insulin tolerance test of WT and and WT of test tolerance insulin and level insulin ) Serum 6 0 3 Body weight (g) −1 = 10 per group ( group per = 10 Body weight (g) –1 –1 0 20 40 60

10 20 30 40 50 IS-GDR (mg kg min ) ) ) by oral gavage. Consistent with the phenotype of the KO 0 ). This compound is highly selective for Ltb4r1 versus versus Ltb4r1 for selective highly is compound This ). 0 10 20 30 2 0 ** 6 9 0 ), and its structure is provided in refs. refs. in provided is structure its and ), advance online publication online advance Vehicle WT Vehicle ** 0 j ) Glucose tolerance test, body weight, and serum serum and weight, body test, tolerance ) Glucose Inhibito e ) Insulin tolerance test of HFD-fed mice treated treated mice HFD-fed of test tolerance ) Insulin 120 ** ** ** KO b Week Week – r e h Inhibitor e ,

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© 2015 Nature America, Inc. All rights reserved. percentage of PKH26 of percentage ( mice. WT obese in ( performed. is inhibitor Ltb4r1 or vehicle with labeled ( line. cell macrophage a human of chemotaxis CM-induced ( IP-macs. of chemotaxis ( chemotaxis. ( vitro in 2 Figure ( mice male the in as effects beneficial comparable observed and inhibitor Ltb4r1 the with mice OVX HFD-fed, treated we Therefore, resistant. insulin become and to male mice, ovariectomized (OVX) female mice gain weight on HFD lower serum insulin and free fatty acid (FFA) levels ( ( weight body in insulin ( glucose tolerance ( mice, treatment with the Ltb4r1 inhibitor resulted in markedly greater nature medicine nature in described is strategy FACS gating expressed as mean mean as expressed epi-WAT from Cd11c e b g f e a ) A schematic diagram showing injection of donor blood monocytes monocytes blood donor of injection showing diagram ) A schematic ) Effect of Ltb4r1 inhibitor (100 nM) on LTB4-induced macrophage macrophage LTB4-induced on nM) (100 inhibitor Ltb4r1 of ) Effect Ltb4r1 Lean mous

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macrophages, and neutrophils in SVCs from vehicle- or inhibitor-treated HFD-fed mice. ( mice. HFD-fed inhibitor-treated or vehicle- from SVCs in neutrophils and macrophages, F4/80 APC 660/20-A ex vivo ex

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Ltb4r1 inhibitor blocks macrophage chemotaxis both both chemotaxis macrophage blocks inhibitor Ltb4r1 –139 0 Basal 2 3 4 5 M –304 in vivo in –1 e c ob/ob 0 ) Effect of Ltb4r1 inhibitor on 3T3-L1 CM-induced CM-induced 3T3-L1 on inhibitor Ltb4r1 of ) Effect with PKH26 dye. Cells are then injected into recipient mice, and and mice, recipient into injected then are Cells dye. PKH26 with e 2 01 ) ) compared to vehicle-treated mice without any change CD11c PE576/26-A Labeled with F4/8 0 Fig. 1 Fig. . . ( LTB4 (nM) 2 Transfe PKH26 ± treated with vehicle or Ltb4r1 inhibitor. Data were analyzed by two-way ANOVA followed by Bonferroni Bonferroni by followed ANOVA two-way by analyzed were Data inhibitor. Ltb4r1 or vehicle with treated 0.1 g Fig. Fig. 1

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© 2015 Nature America, Inc. All rights reserved. vehicle or Ltb4r1 inhibitor. Data were analyzed by two-way ANOVA followed by Bonferroni Bonferroni by followed ANOVA two-way by analyzed were Data inhibitor. Ltb4r1 or vehicle ( inhibitor. Ltb4r1 or vehicle with ( inhibitor. Ltb4r1 or vehicle with treated mice HFD-fed 3 Figure ( treatment inhibitor Ltb4r1 by induced homeostasis tissue adipose of improved to indicative mice, ninefold from changed is ATMs twofold in mice compared the vehicle-treated to the inhibitor-treated M1/M2 of ratio the Indeed, macrophages recruited macrophages of population in the Ltb4r1 number inhibitor–treated Cd11c the of number mice higher a ( is lower there while Cd11c, express even which an is there that noninflammatory are which ATMs, Cd11c-negative M2-like to compared proinflammatory and groups. M1-like are the Cd11c expressing (ATMs) between macrophages tissue Adipose macrophages labeled of subpopulations the vehicle-treated ( to mice compared mice inhibitor–treated Ltb4r1 the in tissue adipose in macrophages labeled of degree lower a Wefound substantially vehicle. or inhibitor Ltb4r1 the with treated mice HFD-fed WT recipient into injected then were monocytes labeled The dye PKH26 fluorescent a with from labeled and mouse obtained donor WT were monocytes circulating approach, this With an using tissue adipose vivo in tissue CM. Toadipose determine if these in chemokine important an is ( LTB4 that indicating toxicity cell without causing U-937 line cell both macrophage of human a and chemotaxis IP-macs mouse CM-induced abolished inhibitor Ltb4r1 The a LTB4. and Cxcl1, contains Ccl2, including (CM) chemokines, different of medium mixture ( adipocyte–conditioned chemotaxis differentiated LTB4-induced blocked inhibitor ( manner a dose-dependent in (IP-macs) macrophages intraperitoneal mouse macrophages, for chemokine potent a neutrophils and is T cells. We found LTB4 that LTB4 stimulated chemotaxis of that known is It Effect of Ltb4r1 inhibition on macrophage chemotaxis a change in body weight, and with lower insulin levels ( glucose tolerance compared to vehicle-treated ( tissues: muscle, liver and fat. Furthermore, to led treatment treatment of greater genetically inhibitor obese Ltb4r1 Thus, sensitivity). insulin tissue ( (HGP) production glucose hepatic to both suppress of insulin ability ( sensitivity) insulin muscle skeletal of enhanced insulin-stimulated glucose disposal rate (IS-GDR, a measure ( (GIR) rate infusion glucose overall higher a by manifested was sensitivity ( mice vehicle-treated A n ob Fig. 1 Fig. a = 6 per group. * group. per 6 = Relative expression s e l c i t r /

(fold induction) ob 0.5 1.0 1.5 ) mice with the Ltb4r1 inhibitor for 2 weeks also led to better to led also weeks 2 for inhibitor Ltb4r1 the with mice ) Fig. 2 Fig. condition, we directly measured macrophage migration into into migration macrophage measured directly we condition, 0 h Fig. 1 Fig.

in vivo in ) and circulating FFA levels ( FFAlevels circulating and ) Ltb4r1 inhibitor improves epi-WAT inflammation in HFD mice. ( mice. HFD in epi-WAT inflammation improves inhibitor Ltb4r1 Fig. 2 Fig. Tnf Inhibitor Vehicle e g ). These data were more revealing when we examined examined we when revealing more were data These ). ). In addition, the inhibitor-treated mice exhibited an an exhibited mice inhibitor-treated the addition, In ). insulin sensitivity in all three major insulin target target insulin major three all in sensitivity insulin a ), and pretreatment of the IP-macs with the Ltb4r1 Ltb4r1 the with IP-macs the of pretreatment and ), P Il6 < 0.05, ** 0.05, < ** Ccl2 i. 1g Fig. i. 2c Fig. in vivo in in vitro Ccl5 P Fig. 2 Fig. 3 < 0.01 for vehicle versus inhibitor. versus vehicle for 0.01 < 2 c . By using FACS analysis, we found found we analysis, FACS using By . – – macrophage-tracking technique macrophage-tracking , f i chemotaxis results translated to the d ) Serum levels of the cytokines Il-6 ( Il-6 cytokines the of levels Serum ) ). This improvement in insulin insulin in improvement This ). Itgax e and and ). Fig. 1 Fig. Fig. 1 Fig. upeetr Fg 3 Fig. Supplementary Relative expression b i ) (a measure of adipose adipose of measure (a ) ob

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) Relative mRNA expression of inflammatory cytokines in epi-WAT of in cytokines inflammatory of expression mRNA Relative ) ** Additionally, in in Ltb4r1 inhibitor–treated mice ( vivo in and chemotaxis IP-mac the with Consistent mice. inhibitor–treated of into for Ltb4r1 tissues of the concept migration substantially lower macrophage was tissue ( adipose cells WT to compared impaired into migrate to monocytes KO of ability the that Wefound mice. obese HFD-fed WT into or mice WT either from monocytes donor KO mice. We found that LTB4 stimulated phosphorylation of Ikk- of phosphorylation LTB4stimulated We that found KOmice. ie oprd o h vhcetetd ns ( ones vehicle-treated the to compared mice Mrc1 including genes, of anti-inflammatory expression ( latter the versus mice former of the group in (epi-WAT) tissue adipose white epididymal in state resistant insulin inflammatory-induced the with associated typically we also found lower expression of a numbermice, of proinflammatory genesvehicle-treated HFD-fed to compared mice Ltb4r1 inhibitor–treated HFD-fed in accumulation ATM lower the with Consistent LTB4 and Ltb4r1 induce inflammation Cd11c Cd11c KO mice ( mice KO but it did not affect the expression of anti-inflammatory genes ( gene expression, as in was observed LTB4 cytokine treatment of IP-macs inflammatory in resulted higher flammatory and anti-inflammatory genes by quantitative PCR (qPCR). we inflammation, measured the mRNA expression of different proin cells. these of pathway inflammatory the in involved are and Jnk and promoted I and WT from IP-macs isolated in stimulation inflammatory proinflammatory pathways. To this ATMend, we evaluated LTB4-mediatedintrinsic activate directly also could LTB4 that possibility the ( blood and and tissue adipose in markers proinflammatory of ( treatment inhibitor Ltb4r1 ( mice vehicle-treated HFD-fed, to compared Tnf, Il-12p40, Ccl5, and Il-6, by exemplified as cytokines, proinflammatory various of levels lower in resulted mice in HFD-fed of elevated treatment are inhibitor and obesity, levels cytokine circulating inflammation, tissue to induced Jnk activation in IP-macs from WT mice but not from compared to vehicle-treated controls. The Ltb4r1 inhibitor blocked LPS- Fig. 3c Fig. We also conducted conducted also We To further demonstrate that LTB4 and Ltb4r1 promote macrophage expression lower the for explanation one is content ATM Lower Mrc1

3 ** d , and and , ) in Ltb4r1 inhibitor–treated mice. In addition, we considered considered we addition, In mice. inhibitor–treated Ltb4r1 in ) ), Tnf ( Tnf ), − + migration results, we found lower amounts of Cd11c of amounts lower found we results, migration ATM ( content ATMs and adipose tissue neutrophils (ATNs) but to higher higher to but (ATNs) neutrophils tissue adipose and ATMs – f Il4 ). Epi-WAT mass and adipocyte size were not changed by changed not were size adipocyte Epi-WAT and ). mass Fig. 4 Fig. * Il4 post hoc post e ) and Il-12p40 ( Il-12p40 and ) , was observed in epi-WAT from the inhibitor-treated inhibitor-treated the epi-WAT in from observed was , advance online publication online advance ob/ob c

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© 2015 Nature America, Inc. All rights reserved. in in * experiments. independent three than more of representative n mean as expressed are Values tests. Bonferroni by followed ANOVA two-way by analyzed were Data analysis. qPCR or WT Nos2 on nM) (100 inhibitor ( IP-macs. in LPS by induced p-Jnk on nM) (100 inhibitor Ltb4r1 of ( LTB4 after stimulation. IP-macs ( ( LTB4or LPS stimulation. after IP-Macs in p-Jnk ( inflammation. 4 Figure as (pJnk), phosphorylation Jnk on LTB4 of nism ( mecha intervening likely the is activation shown). not In contrast, our (data data indicate that Jnk signaling insulin downstream in steps subsequent or phosphorylation serine of LTB4 on Irs1 effects the not block did tors siRNA knockdown or inhibi small-molecule Ikk- of inhibition that found we studies, In our Irs1. target can kinases serine specific that known well is it and signaling, downstream its impair can phosphorylation ( phosphorylation Akt insulin-stimulated of LTB4 inhibition of the on effects the blocked treatment PT addition, In actions. LTB4-mediated these ( port of LTB4trans effect the glucose to blocked inhibit pretreatment this G the with cells G with couple can signaling As Ltb4r1 in myocytes. 5c ( Fig. cells these in translocation (Glut4) 4 type porter ( rylation phospho Akt insulin-stimulated reduced by accompanied is effect ( ~50% inhibits by transport directly glucose it that insulin-stimulated found and myocytes L6 on LTB4 of effect ( adipocytes 3T3-L1 in not that ( mice obese HFD-fed from and liver, adipose muscle, tissues, target are insulin levels major LTB4 three all in function. elevated of loss Ltb4r1 to due effects sensitizing or inhibitor effects Ltb4r1 the anti-inflammatory of the and sensitivity, insulin reduced for mechanism is a key inflammation tissue chronic Obesity-associated LTB4 directly induces cellular insulin resistance in had no effect IP-macs to those in observed WT in responses LPS the lowered inhibitor Ltb4r1 the and cells, WT in lower was expression gene matory inflam LPS-stimulated Further, responses. inflammatory in system nature medicine nature in brackets b b = 6 per group ( group = 6 per ) or anti-inflammatory M2-like ( M2-like anti-inflammatory ) or , Insulin receptor substrate 1 (Irs1) serine serine (Irs1) 1 substrate receptor Insulin b c or for the comparisons indicated by the the by indicated comparisons the for or ) qPCR analysis of proinflammatory M1-like M1-like proinflammatory of analysis ) qPCR Ltb4r1 ( Fig. 5 Fig. Ltb4r1 h Fig. Fig. 5c ), consistent with direct interference with insulin signaling signaling insulin with interference direct with consistent ), ), ),

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0 (fold induction) ). This LTB4 This ). 0.5 1.0 1.5 2.0 – – – 0 – – – – + – WT + + – WT Arg1 + + – * LTB4 Vehicle + + – * + + – + * Nos2 Tnf + * - - - - +

– – Ltb4r1 – – Ltb4r1 Il10 + – not inhibited by insulin, consistent with an LTB4-induced cellular cellular LTB4-induced an with consistent insulin, by inhibited not ( affect not did but HGO stimulated directly ( insulin by inhibited lates hepatic glucose output (HGO) and that this effect was completely cells. L6 in transport glucose to Akt through signaling downstream decreased with phosphorylation serine Irs1 subsequent to leading thereby activation, Jnk promotes a through G resistance insulin cellular LTB4 that promotes view the with are consistent data these together, ( myocytes L6 in resistance insulin cellular LTB4-induced on depletion siRNA as effects same the had of inhibitor Jnk and Jnk inhibition found that activity pharmacologic ( LTB4 treatment by tion phosphoryla of Irs1 and serine augmentation it prevents completely phosphorylation, Akt insulin-stimulated of inhibition on LTB4 of ( LTB4 treatment on inhibition of transport glucose insulin-stimulated L6 with myocytes siRNA to is sufficient completely block ofthe effect cells or cells treated with insulin alone ( untreated to compared levels pJnk higher promotes concentrations different two at LTB4 that found and stimulation, Jnk of readout a insulin resistant state. Further, LTB4 treatment blocked the effect of effect the blocked LTB4 treatment Further, state. resistant insulin + – Fig. Fig. 5 Fig. 5 Fig. + + b + – In WT primary hepatocytes KO + + + – KO Relative expression (fold induction) 0.5 1.0 1.5 2.0 2.5 e d Mgl1 ). This effect of ). LTB4effect This of on HGO stimulation in was cells WT 0 Inhibitor ). Consistent with this, Jnk depletion also blocks the effect effect the blocks also depletion Jnk this, with Consistent ). i Inhibitor f Relative expression Relative expression LPS

(fold induction) LPS

(fold induction) Tnf LTB4 Vehicle 0 1 2 3 4 0 1 2 3 4 5 * Clec7a – – – – + – WT + – WT + + – * Fig. 5 Fig. Il6 + + – * Mrc1 * + * + Fig. 5 Fig. * Il1b Il6 – – – – Ltb4r1 e Ltb4r1 ). LTB4 treatment of WT hepatocytes hepatocytes WT of treatment LTB4 ). + – in vitro + – d Ccl2 ). Finally, we used a small-molecule small-molecule a used we Finally, ). + + + – Il4 KO + + + – * KO Supplementary Fig. 7 Fig. Supplementary , , we found that glucagon stimu α Fig. 5 Inhibitor Inhibitor g j i-coupled mechanism, which which mechanism, i-coupled Inhibitor Relative expression Relative expression d Il1b LPS LPS (fold induction) (fold induction) p-Jnk LPS Jnk 0 2 4 6 8 0 3 6 9 c Ltb4r1 ). ). Depletion of Jnk from – – – – – – – – + + – + – WT WT Nos2 s e l c i t r a WT -KO hepatocytes hepatocytes -KO + + + – * + + – * + + + Cxcl1 + Ccl2 * * – – – – – – – – Ltb4r1 Ltb4r1 Ltb4r1 Cxcl1 + – + – ). Taken ). + + + + – + + + – KO + + KO KO + - - © 2015 Nature America, Inc. All rights reserved. ANOVA followed by Bonferroni Bonferroni by followed ANOVA two-way by analyzed were Data mice. inhibitor–treated Ltb4r1 or vehicle in (right) concentration (Ins) insulin and (middle), test tolerance glucose ( ( hepatocytes. and WT in output glucose on insulin and glucagon ( cells. L6 in (Ser307) p-Irs1 and p-Akt uptake, glucose stimulated ( ml ng (100 PT and ( cells. L6 in p-Akt and uptake glucose stimulated insulin- on ( cells. 3T3-L1 and hepatocytes and ( hepatocytes. and myocytes in resistance 5 Figure ylation in LTB4 had no oneffect the ability of insulin to promote Akt phosphor LTB4 ( with treatment concomitant by inhibited in WT primary hepatocytes was quite potent, this effect was completely unknown. is this for robust in 8a Fig. be completely blocked by Ltb4r1 inhibitor treatment ( lism. Showing the of specificity this effect, LTB4-stimulated HGO can the normal inhibitor effect of insulin on this aspect of hepatic metabo attenuate also can and itself by HGO promote can LTB4 stimulation ( cells KO in effect inhibitory without was but cytes, hepato WT in HGO glucagon-stimulated of inhibition on insulin A experiments. independent three than more of LTB4 (30nM) a f i d b a d p-Akt (S473)

) Effect of Jnk siRNA on glucose output in primary hepatocytes. ( hepatocytes. primary in output glucose on siRNA Jnk of ) Effect ) Relative mRNA expression of of expression mRNA ) Relative ) Effect of LTB4 and Jnk siRNA on insulin insulin on LTB4 of siRNA Jnk and ) Effect LTB4 of G the and ) Effect

Hsp90 Glucose uptake Relative mRNA expression Ins (10nM) We also found that although insulin-stimulated Akt phosphorylation –1 s e l c i t r 0.0 0.0 0.0 0.0 0.1

Ltb4r2 (cpm mg protein, ×1,000) Jn 10 0 2 4 6 8 2 4 6 8 0 k 80 20 40 60 0 p-Akt/Akt 0 ). The overall effect of glucagon on increasing HGO was less less was HGO increasing on glucagon of effect overall The ). Ak

0 1 2 3 4 5 LTB4 directly induces insulin insulin induces LTB4 directly t Ltb4r1 Ltb4r1 Ctrl-siRNA in L6 cells, primary primary cells, L6 in + – + – – – Basal ** L6 g WT ) Effect of LTB4 on p-Irs2 (Ser307) in primary hepatocytes. ( hepatocytes. primary in (Ser307) LTB4of p-Irs2 on ) Effect ** + + -KO cells compared to WT cells -KO ( to compared cells WT cells -KO hepatocytes ( −1 Ins Hepato- ) on p-Jnk in L6 cells. cells. L6 in p-Jnk ) on cyte Jnk-siRNA ** e c ) Effect of LTB4,of ) Effect ) Effect of LTB4of ) Effect p-Akt/Akt Ins +LTB4 0 1 2 3 4 5 α + – + – – – adipocyte Ltb4r2 Ltb4r1 3T3-L i inhibitor PT i inhibitor post hoc post

KO LTB4 (100nM

Ltb4r1 + + Ins (100nM p-Akt (S473) 1 Fig. Fig. 5 Tubulin siRN PT (100ngml b

g pSer307-Irs2 LTB4 (100nM) tests. Values are expressed as mean mean as expressed are Values tests.

LTB4 (nM Ins (100nM) C A ) ) f

). ). Similar to the results in L6

p-Irs2/Irs2 Acti Glucose uptake – – ## Irs2 trl –1 0.5 1.0 1.5 2.0 2.5 (cpm mg protein, ×1,000) 0 n P – ) 10 20 30 Ctrl –1 + < 0.01 for KO versus WT in in WT versus KO for < 0.01 – 0 – ) Ltb4r1 j Ctrl Fig. 5 Fig.

–1 + + Fig. 5 Fig. Blood glucose (mg dl ) – – ** 100 200 300 400 30 ** Jnk + + + Supplementary Supplementary – – -KO primary hepatocytes. ( hepatocytes. primary -KO Fig. 5 Fig. f ). In contrast, contrast, In ). p-Irs1 (Ser307) e LTB4 (100nM ** 100 + – – 3 0 ** ); the reason ); the reason Ins (100nM) + + + – e h ). Thus, Thus, ). Glucagon (10ngml Hsp90 SiRNA j 6 0 ) Effect of clodronate on glucose tolerance (left), area under the curve (AUC) of (AUC) curve the under area (left), tolerance glucose on clodronate of ) Effect – – – Time (min) Irs1 ** PT (100ngml ) + + 9 0 LTB4 (30nM) 14C glucose production - - - Ctrl

–1 + + + (cpm mg protein, – – h ± Inhibitor-clodronat Veh-clodronat Inhibitor-PBS Veh-PBS 0 ×1,000) ) Effect of PT (100 ng ml ng (100 PT of ) Effect sem. sem. is not expressed in these cells ( cells these in expressed not is 8c Fig. ( adipocytes 3T3-L1 in transport glucose stimulated insulin- inhibit not did LTB4 hepatocytes, and myocytes in action resistance insulin in terms of HGO suppression ( LTB4hepatocyte of on effects the blocked Jnk of depletion mediated phosphorylation ( and it also blocked the ability of LTB4 to inhibit insulin-stimulated ( Akt HGO glucagon-stimulated of inhibition insulin-mediated by the fact that PT treatment prevented the effects of LTB4 on blocking insulin resistance are mediated through G rylation ( myocytes, LTB4 treatment led to Irs2 hepatocyte 307 serine phospho e 100 150 200 50 , , * Insulin stimulated –1 –1 In contrast to the effects of the LTB4–Ltb4r1 system on insulin insulin on system LTB4–Ltb4r1 the of effects the to contrast In 120 Ctrl glucose uptake (% of INS) P ) ) + – 100 150 50 < 0.05, ** < 0.05, n 0 f – – – = 6 per group ( group = 6 per ) Effect of LTB4 on p-Akt in WT and and WT LTB4in of p-Akt on ) Effect ). This is fully consistent with the results showing that that showing results the with consistent fully is This ). ** e In Fig. Fig. 5 Ctrl + + ** I s + – – – e LTB4 ns ** + + –

–1 g P advance online publication online advance

AUC (mg dl min) Jn p-Akt (S473) ). ). These effects of LTB4 that directly cause hepatocyte < 0.01 in in < 0.01 LTB4 (100nM) + + 10,000 15,000 Supplementary Fig. 8b k 5,000 Ins (100nM) + + – – ** 0 a

LTB4 (30nM) p-Akt/Akt , Ak + – b −1 (10 ngml 0. 1. 1. 2. Ins (10nM) e , t 5 0 5 0 0 Glucagon 14 d ) on glucose output in primary hepatocytes. hepatocytes. primary in output glucose ) on

PBS C glucose production , –1 b e + + – (cpm mg protein, ×1,000) , , – – – – h 100 150 200 d ** 50 –1 – , i 0 e ) j ** Clodronate Fig. 5 Fig. ). Western blot data are representative representative are data blot Western ). + , h 14C glucose production – –1 j + + – – – . (cpm mg protein, ×1,000) 20 40 60 ** Inhibitor Vehicl 0 a + ). Ltb4r1 ). As with the L6 cells, siRNA- α e + – – – Ins (100nM) c ## i signaling, as demonstrated Basa ** ** LTB4 (nM) Ctrl-siRNA ** Hsp90 ** p-Jnk G l –1 ** + + -KO primary primary -KO Ins (ng ml ) Jn ##

** 10 15 k Fig. Fig. 5 0 5 lucagon nature medicine nature – – + + – – – Supplementary Supplementary ## PBS + – i ). 30 – ** ** LTB4 + Jnk-siRNA 100 ## Clodronate * – Fig. 5 Fig. Ltb4r1 30 Inhibitor Vehicl + * + + + ## 100

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© 2015 Nature America, Inc. All rights reserved. ih T4 oh tmltd nlmaoy ahas n macro in pathways hepatocytes in resistance insulin inflammatory cellular caused directly and phages stimulated both LTB4 with Ltb4r1 inhibitor–treated animals versus controls. in Inmethod, clamp contrast, treatment hyperinsulinemic-euglycemic the by measured as observed better glucose tolerance, we as welladdition, In as systemic mice. insulin sensitivity vehicle-treated to compared steatosis hepatic and levels, less pathway cytokine lower genes, blood of inflammatory lower expression tissue, in adipose by accumulation less macrophage This obesity. robust phenotype genetic-mediated in the Ltb4r1 inhibitor–treated and mice was manifested HFD-induced both of the in setting resistance insulin systemic from protection with phenotype Here we show that inhibition of Ltb4r1 results in an anti-inflammatory DISCUSSION resistance. insulin hepatic mediated vivo in insulin action ( augmented genes lipogenic ( and HFD-fed both in absent largely was mulation sis to hepatosteato marked leads obesity It is that known HFD-induced Hepatic lipid profiles cells. target insulin on effect sensitizing insulin- direct a and cells immune in effect anti-inflammatory an of combination a through mediated are inhibitor Ltb4r1 the of effects 1 Fig. than the glucose and insulin levels in chow-fed mice ( ( levels insulin plasma of lowering ~60% further a with group vehicle/clodronate-treated the than tolerant glucose more ~40% were inhibitor Ltb4r1 the and nate greatly improve insulin resistance. However, mice treated with clodro ( HFD mice, and this is accompanied by lower circulating insulin levels glucose tolerance in HFD-fed mice compared to vehicle (PBS)-treated better with associated is injection clodronate the findings, previous Ltb4r1 inhibitor–treated mice by clodronate injection. Consistent with resistance insulin ( inhibitor. Ltb4r1 or vehicle with treated bar, 100 Scale inhibitor. Ltb4r1 or vehicle 6 Figure nature medicine nature myocytes. and inhibitor or LTB4 versus vehicle (PBS). LTB4vehicle or versus inhibitor Bonferroni by followed ANOVA two-way by analyzed were Data a Fig. 6c Fig. Fig. 5 Fig. 6 Fig. In primary hepatocytes, we found that LTB4 treatment directly directly treatment LTB4 that found we hepatocytes, primary In on LTB4 of effects different these of contribution the dissect To

33 ob/ob mice HFD mice 100 100 , µ µ 3 m m 4 ). These results indicate that the the that indicate results These ). j . After 2 weeks of Ltb4r1 inhibitor treatment, hepatic fat accu hepatic treatment, . 2 of inhibitor After weeks Ltb4r1 , thereby providing a possible further mechanism for LTB4- for mechanism further possible a providing thereby , a ), ), showing that of depletion macrophages alone is to sufficient ,

d ), and this was accompanied by reduced expression of the the of expression reduced by accompanied was this and ), Ltb4r1 inhibitor improves hepatic steatosis. ( steatosis. hepatic improves inhibitor Ltb4r1 Vehicle ). ). Because ceramide and DAGs have been reported to inhibit e novo de 35– Nr1h3 3 9 , , LTB4 might induce higher amounts of these lipids in vivo in advance online publication online advance synthesis of hepatic DAGs and ceramides ceramides and DAGs hepatic of synthesis 100 , , µ m Srebf1 , we depleted macrophages in control and and control in macrophages depleted we , Fig. 5 Fig. Inhibitor and and j ), and these values are even lower lower even are values these and ), Ldlr c , ( d µ Fig. 6 Fig. ) Effect of LTB4 on DAG content ( LTB4of content DAG on ) Effect m. ( m. n vivo in Relative expression b

b (fold induction) ) Relative mRNA expression of lipogenesis and gluconeogenesis genes in livers of HFD-fed mice mice HFD-fed of livers in genes gluconeogenesis and lipogenesis of expression mRNA ) Relative 0.5 1.0 1.5 2.0 b 0 ). insulin-sensitizing insulin-sensitizing a Scd1 ob ) Representative images ( images ) Representative Supplementary Supplementary

/ ob obese mice mice obese Fasn post hoc post Ldlr tests. Values are expressed as mean mean as expressed are Values tests. ** - - - -

Nr1h3 c its direct effects on cellular insulin resistance in hepatocytes and inflammation– and the to understanding hepatocytes of layer in new a adds resistance myocytes, insulin cellular on effects direct its all can cells that target LTB4,produce LTB4’s insulin along with Given on promoting inflammation, effect and cells resistance. immune other insulin macrophages, and inflammation tissue chronic between connection the reinforces treatment inhibitor Ltb4r1 with of insulin resistance is independent of effects of the LTB4–Ltb4r1 LTB4–Ltb4r1 the of effects of independent is resistance component insulin of This cells. liver and muscle in resistance insulin cause ( mice HFD from in tissues LTB4 concentrations higher for the mechanism a potential obesity in reported been have FLAP and AA of generation of LTB4 from arachidonic acid (AA) and the elevated levels tissue in obesity adipose in LTB4 levels higher showing studies other with consistent proinflammatory major the all pathways and of is in elevated the three key target insulin This tissues. is downstream is LTB4 because resistance, insulin inflammation-induced for mechanism new a be could This response. inflammatory tissue chronic the propagating further LTB4, more produce then cells immune these ATMs; vates tissue cells such produce LTB4,as adipocytes and which recruits acti Thus, cycle. vicious positive-reinforcing a up setting LTB4, produce sensitivity insulin and tolerance glucose improved with along in phenotype an anti-inflammatory show studies recent with consistent is This syndrome. resistance inflammation–insulin ultimate the inhibiting tissue, adipose into cells immune other and monocytes of tracking HFD and the and migration into tissues. Strong support for this notion comes from chemotaxis cause to types, cell immune on Ltb4r1 through working chemokine, powerful a as acts LTB4 obesity. in state inflammatory resident tissue cell types could be an early trigger for the chronic tissue or cells, other endothelial myocytes, hepatocytes, such as adipocytes, cells tissue primary by LTB4 of production suggest studies obesity-associated that These loss- Ltb4r1. at specific only of directed studies representative of-function are inhibitor Ltb4r1 with treated Ltb4r2 neutrophils and Ltb4r1 through (refs. operating factor chemotatic potent syndrome. resistance insulin ) and ceramides ( ceramides ) and This improvement in inflammatory status and insulin sensitivity sensitivity insulin and status inflammatory in improvement This One of the novel findings in this study is that LTB4 can directly directly can LTB4 that is study this in findings novel the of One also can cells immune recruited that note to important is It a is LTB4 that showing literature of body substantial a is There n Srebf1 in vitro = 6) of H&E staining of liver from HFD or or HFD from liver of staining H&E of = 6) 40 ** , 4 Inhibito Vehicl Acaca ob 1 chemotaxis and ). Ltb4r1 is more specifically distributed on macrophages, distributed ). is Ltb4r1 more specifically 4 / 2 ob and lymphocytes and e r mice. Thus, Ltb4r1 inhibition impairs chemotaxis and 4 4 d c Fig. Fig. . 5-LO–activating . protein 5-LO–activating plays (FLAP) a role in the ) in primary hepatocytes from C57BL6/J mice mice C57BL6/J from hepatocytes primary ) in DAG (% of total lipids) 10 0 2 4 6 8 1 ). 30 min in vivo ± ** sem. sem. 4 2 LTB4 Ins Vehicle 3 5 , and both the KO mice and mice mice and mice KO the both and , monocyte tracking experiments in experiments tracking monocyte . n 2 = 6. ** = 6. h ** d P ob

Ltb4r1 Ceramides (cpm/mg protein) < 0.01 for vehicle versus versus vehicle for < 0.01 10,000 15,000 / 5,000 ob 44– mice treated with with treated mice 0 s e l c i t r a -KO mice, which which mice, -KO 4 6 , thus providing providing thus , 30 min LTB4 In Vehicle ** s in vitro in 2 h ** . -

© 2015 Nature America, Inc. All rights reserved. the treatment of insulin-resistance diseases. insulin-resistance of treatment the in goal of LTB4inhibition future therapeutic be a could useful action that possible is it such, As sensitivity. insulin impaired and mation inflam connecting mechanism new a provides resistance insulin of mechanism cytokine-independent This myocytes. and hepatocytes Thus, resistance. insulin LTB4-induced for mechanism additional novel a reveal also studies Our pathways. proinflammatory vate intracellular into adipose tissue and directly stimulates macrophage Ltb4r1 to acti monocytes of migration promotes that chemokine potent a is LTB4 a is system LTB4–Ltb4r1 majorthe driver for that the inflammation–insulin resistanceand syndrome. Thus, obesity in tissues target lin efficacious. more be resistance and that attempts insulin to inhibit the for LTB4–Ltb4r1 system might mechanisms key the at directed fully not are modalities therapeutic these that possibility the raise can one studies, current sensitivity insulin improved not tions, effects only had has salsalate effects modest agent anti-inflammatory general more the studies clinical in sensitivity insulin improving on effect TNF- cific spe of effects glucose-lowering shown have studies some Whereas success have had limited resistance of insulin inflammation-induced cause key a is inflammation resistance insulin tissue chronic that years several action insulin in defects to contributing resistance. insulin and cellular cause LTB4 that of effects direct inflammation the with interfering obesity-induced attenuating both by signaling Ltb4r1 of blockade that view the with consistent is This respectively. levels, or insulin glucose in reduction further 40–60% a to led treatment inhibitor Ltb4r1 plus clodronate whereas hyperinsulinemia, and tolerance glucose in improvement marked a caused mice clodronate-treated in depletion macrophage myocyte and hepatocyte insulin sensitivity are operative resistance. insulin cellular induced reasonable G Taken and a L6 provide myocytes. results together, these hepatocytes in isolated resistance insulin of LTB4 cellular to effects induce the all inhibits of Jnk function loss pharmacologic and genetic Both events. activity, mediating the inhibitory Irs1 kinase and Irs2Jnk serine phosphorylation stimulated LTB4 that found we Furthermore, signaling. G of insulin and steps proximal with to interfere myocytes and Ltb4r1 hepatocytes through specifically works LTB4 studies that our indicate resistance, insulin induce to LTB4 of effects direct the of mechanisms to respect With response. inflammatory tissue pose adi the of attenuation to due mainly is treatment inhibitor Ltb4r1 by conferred sensitivity insulin tissue adipose the that conclude we tissues, target insulin major three all in resistance insulin systemic demonstrate studies clamp hyperinsulinemic-euglycemic the Since and myocytes but hepatocytes, not as in the adipocytes, latter both do not express Ltb4r1. in operates mechanism this demonstrated, As resistance. insulin of mechanism this direct more a sense as at certain looked a be can in response, inflammatory an of part is of LTB4 production Although inflammation. tissue promote to pathway A

In summary, results these show that LTB4 is overexpressed in insu mechanisms molecular multiple probably are there Although of LTB4 effects direct reduced that causing these Our data indicate s e l c i t r 57 in vitro in , 5 8 α , but these have been ascribed to enhanced beta cell func cell beta enhanced to ascribed been have these but , or IL-1 α , LTB4 directly caused reduced insulin sensitivity in in sensitivity insulin reduced caused directly LTB4 , 49 i- i- and mechanism for Jnk-dependent molecular LTB4- , 54– 4 8 5 β . However, therapeutic efforts to ameliorate this this ameliorate to efforts therapeutic However, . 6 inhibitors . . IL-1 β inhibition does lead to glucose-lowering to lead does inhibition glucose-lowering 50 , 5 1 , these , agents have these had to little no in vivo in 50 4 , 57 7 , it has been known for for known been has it , , 5 8 . On the basis of the the of basis the On . can improve insulin insulin improve can in vivo 49– 5 3 . . Thus, , Even Even , α i in in i 4 9 ------.

project, and co-wrote the manuscript. genotyping; P.L. and J.M. Olefsky analyzed and interpreted the the data, supervised in the GTT female ovariectomized mice; J.M. Ofrecio and S. Taguchi assisted with assisted with tissues collecting and gene expression measurements; H.C. performed assisted with clamps;hyperinsulinemic-euglycemic A.J., M.M., O.O. and R.M. uptake and gluconeogenesis assay and western blot. W.S.L., M.L. and S. Talukdar performed macrophage chemotaxis, signaling, and FACS analysis; G.B. did glucose P.L. the designed studies and performed most of the experiments; D.Y.O. (J.M. Olefsky), and a grant to J.M.Olefsky from Merck, Inc. Cooperative Centers Program in Reproduction and Infertility Research of HealthChild and Human Development/National Institutes of Health through a DK074868, DK063491, DK09062), the Eunice Kennedy Shriver National Institute US National Institute of Diabetes and Digestive and (DK033651, Kidney Diseases L6 myocytes. This study was funded in part by grants to J.M. Olefsky from the mice. We thank A. Klip at The Hospital for Sick Children in Canada for providing We thank A.D. Luster from Harvard Medical School for providing the online version of the pape Note: Any Supplementary Information and Source Data files are available in the the of version in available are references associated any and Methods M 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial A AUTHOR CO C c omp

ethods

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© 2015 Nature America, Inc. All rights reserved. kit (STEMCELL) following the manufacturer’s instructions. enrichment Isolated monocyte monocytesmouse EasySep with enriched were subsets monocyte and buffer lysis RBC to subjected were sinus, retro-orbital the by bled mice, In vivo 2-phenylindole and counted. 3 h of migration, cells were fixed in formalin and stained with 4 DMEM with LTB4 (Cayman Chemical) was placed in the lower chamber. After or medium conditioned adipocyte differentiated 3T3L1 min; 30 for inhibitor 8 macrophages (IP-macs) or U-937 cells were placed in the upper chamber of an were isolated from mice. ForWT the migration ously described Invitro ies were from BD Biosciences. Serotec (Raleigh, NC); FITC–Cd11b, PE–Cd11c and APC–Cy7–Ly6G antibod IgGs for 30 atmin FACS 4 F4/80–APC °C. antibody was purchased from AbD at 4 °C before staining with fluorescently labeled primary antibodies or control 1% BSA PBS. SVCs were incubated with Fc Block (BD forBiosciences) 20 min (300 centrifugation another by followed min 5 for (eBioscience) buffer lysis (RBC) cell blood red with incubated was SVCs Aldrich) for 30 min. After centrifugation at 500 BSA/PBS. Tissue suspensions were digested with collagenase (1 mg ml 1% in minced then and PBS, in times three rinsed wereweighed, fatpads mal described previously as performed were FACS analyses FACS and analyses. SVCisolation Luminex assay (Millipore/Linco Research). multiplex a using measured were concentration TnfIL-12p40 and Ccl5, IL-6, enzymatically using a kit (HR seriesmeasured NEFA-HR)were levels FFA Plasma from WAKO(ALPCO). kits ELISA Chemicals. with Serum measured was of 50 mg kg 12 of the HFD, received either vehicle or Ltb4r1 inhibitor for 2 weeks at a dose a high fat diet (HFD, 60% kcals from fat) for 14 weeks, and then, starting at week Phenotypic evaluation of mice. described previously as performed were clamps Glucose injection. insulin after min 90 and 60 30, 15, 0, at drawn was blood and fasting, of h 6 after injected IP was kg units 0.5 (ITTs), test tolerance lin insu For injection. dextrose after min 120 and 60 30, 15, 0, at glucose blood kg g (1 erance tests (GTTs), animals were intraperitoneally (IP) injected with dextrose ITTs, GTTs, clamp study.euglycemic and hyperinsulinemic LTB4 measurement was performed as previously described collected from HFD and age-matched normal chow mice. In each group, epi-WAT)and muscle (liver, tissues quadriceps then and age, of weeks were 8 Tissue LTB4 measurement. gavaged at the dose of 50 mg kg ( was orally gavaged daily for 2 weeks atbeginning week 12 of the HFD regimen kg mg (50 inhibitor Ltb4r1 the and weeks 14 for fat) from kcal (60% HFD refs. providedin is structure (ref. Ltb4r2 comparedto Ltb4r1for specific highly compoundis this inhibitor. Ltb4r1 used. when cohorts to assigned randomly were animals all and Animals Laboratory of Use and Care the for Guidelines Research Diego were procedures done animal San with in University accordance of California animals All water. and in mice OVX the female except mice were male food to access free with cycle light-dark 12-h/12-h use. and care Animal ONLINE METHODS nature medicine nature n µ = 10 in each group). each in 10 = m polycarbonate filter (24-Transwell format; Corning) with or without Ltb4r1 −1 monocyte tracking. chemotaxis assay. chemotaxis , Hospira, Inc.) after 6 h of fasting, and blood was drawn to measure to drawn was blood and fasting, of h 6 after Inc.)Hospira, , 45 , −1 60 once a day by oral gavage ( , 6 1 6 . 2 . Briefly, mouse thioglycollate-elicited peritoneal macrophages The Ltb4r1 inhibitor, CP-105696, was provided by Pfizer; Pfizer; by provided was CP-105696, inhibitor, Ltb4r1 The nml wr hue i a aia fclt o a on facility animal an in housed were Animals Ob

/ Invitro C57BL/6J mice were put on HFD for 14 weeks from Leukocyte pools from C57BL/6 WT or ob 2 9 mice (6 weeks old, weeks (6 mice −1 and Male C57BL6/J mice at 8 weeks of age were fed daily for 2 weeks. chemotaxis assay was performed as previ as performed was assay chemotaxis Stromal vascular cell (SVC) isolation and isolation (SVC) cell vascular Stromal 3 −1 0 . 8-week-old mice (C57BL6/J) were fed (C57BL6/J) mice 8-week-old . of insulin (Novolin R, Novo Nordisk)Novo R, (Novolin insulin of n = 10 in each group). Plasma insulin g for 5 min, the pellet containing g per per se Supplementary Figure 2 Figure Supplementary , 5 min) and resuspension in in resuspension and min) 5 , n = 10 in each group) were group) each in 10 = , 100,000 , intraperitoneal100,000 45 , 4 5 6 9 . Briefly, epididy Briefly, . . For tolglucose ′ , , 6-diamidino- Ltb4r1 −1 2 , Sigma- 6 ). The The ). n -KO = 6. . . All −1 - - - - - )

α cells. muscle L6 in uptake Glucose were purchased from ATCC and were negative for mycoplasma. from Sigma-Aldrich, LTB4 was purchased from Cayman Chemical. U-937 cells treated with LPS (100 ng ml treated with either vehicle (DMSO) or 100 nM of Ltb4r1 inhibitor for 1 h, then LTB4 (100 nM) for 6 h. For inflammatory signaling experiments, cells were pre Ltb4r1 inhibitor (10 medium containing DMEM with low glucose overnight, treated with or without previously described as cultured and obtained were macrophages marrow–derived bone and phages reagents. and culture Cell fat tissue and analyzed by FACS. groupeach of mice. Five days SVCs after injection, were isolated from visceral were suspended in 0.2 ml PBS and intravenously (IV) to labeling with PKH26, the monocytes were counted and ~0.5 × 10 were washed once and resuspended in containingserum medium. Subsequent medium supplemented with 10% FBS. The mixture was centrifuged and the cells dark. The staining reaction was stopped by addition of an equal volume (2 ml) ofand mixed, and the cells were incubated for 10 min at room temperature in the ×10 at 2 Two kit). (Sigma-Aldrich) PKH26 of ml labeling PKH26 the in (included C solutionDiluentof ml 2 in suspended and 10 × (2 natants were transferred to a fresh set of tubes and assayed for radiolabeled radiolabeled for assayed and tubes of set fresh a to transferred were natants suspensions to each tube followed by 0.5 ml water. After centrifugation, super microcentrifuge tubes. We added 0.25 ml 5% ZnSO 1.7-ml to transferred were buffers incubation, of end Atthe h. 3 for substrate additional 30 min. These pre-incubations were followed by incubation with the additional 30 min, LTB4 for first 30 min and then with insulin and glucagon for (10 ng ml ethanol), LTB4(0.1% for nM (30 for nM (10 insulin 1 h), or min) 30 glucagonvehicle with h 1 for substrate) of absence (in pre-incubated cells/ were million Cells well. 0.25 containing plates 24–well in buffer ml 0.5 in out carried 0.5 mM, (2 LTB4, orinsulin glucagon and substrates a CO in 5% containing 0.2% fatty acid free BSA, and incubated in the same buffer containing KH salt-bicarbonate (HPSB) buffer (10 mM same mediumHEPES, without serum. Cultures4 weremM thenthe in washedKCl, overnight in cultured HEPES and phosphate-125 nM) (10 dexamethasone mM and FBS NaCl, 10% biotics, 0.85 mM plates in Medium E with fortified non-essential amino acids, GlutaMAX, anti collagen-coated on h attachfor 6 to wereallowed hepatocytes mouse Primary (2400 centrifugation at by buffer in 70 centrifuging times five washed and collected were hepatocytes excised, were livers digested The 10 min followed by a collagenase solutionfor 7.4) (Liberase (pH TM buffer HEPES–phosphate from free Roche) calcium a for with 10 cava vena min. inferior hepatocytes. mouse in outputassay Glucose HCl, mixed with scintillation fluid and counted for radioactivity. N 1 with neutralized vials, scintillation to transferred were extracts the of rest dissolved in 1 N NaOH and an aliquot was used for protein estimation and the radiolabeled culture buffer of followed aspiration by washingby twice withterminated cold were PBS. Cells Incubations were incubation. of min 10 last the 0.4 nM, (100 [3H]-2-deoxy-glucose to exposed were cultures All min. 30 additional an for incubated and added was insulin later, min 15 and, LTB4 with started treatment cultures, of set one In min. 30 for insulin were treated with vehicle (0.1% ethanol) KH or mM 100 nM LTB4 0.83 for NaCl, 45 min and mM Na 100 nM 125 KCl, mM 4 HEPES, mM 10 containing buffer salt HEPES–phosphate to switched then and BSA free acid experiment,of day cultures the were On serum experiments. starved for for used 6 were h in cultures d, 8–10 After formation. myotube and differentiation allow to FBS 2% to switched was medium ency, -MEM -MEM with 10% FBS and antibiotics. After cultures reached conflu 80–90% 2 2 HPO PO 6 4 to 5 × 10 × 5 to , 1.25 mM Na 4 −1 , 1 mM MgSO for 30 min), LTB4 for the first 30 min and then with insulin for an µ Ci Pyruvate/incubation) was used as substrate.Incubations wereas used was Pyruvate/incubation) Ci 6 ) were washed once in serum–free medium (RPMI–1640) (RPMI–1640) medium serum–free in once washed were ) µ g M) for 1 h, and then stimulated with LPS (100 ng ml 2 for 10 min) over a Percoll density gradient (1.06 g/ml). g/ml). (1.06 gradient density Percoll a over min) 10 for HPO 4 6 , 1mM CaCl 3 . For gene expression studies, cells were cultured in in cultured were cells studies, expression gene For . −1 4 Mouse thioglycollate-elicited peritoneal macro peritoneal thioglycollate-elicited Mouse , 1 mM MgCl ) or LTB4 (100 nM) for 45 min. LPS was purchased L6 rat skeletal myoblasts in werecultured rat skeletal L6 g 2 and 0.2% fatty acid free BSA, pH 7.4. Cells for 5 min. Cells were further purified by purified were further for Cells min. 5 2 , 1 mM CaCl Mice were infused through the the through infused were Mice α −3 4 -MEM containing 0.2% fatty M in Diluent C was added added was C Diluent in M and 0.25 ml 0.3 N Ba(OH) - 2 injected retro-orbitally in incubator. 2 and 15 mM NaHCO doi: 10.1038/nm.3800 2 PO µ 14 Ci ml Ci 4 6 , 1.27 mM mM 1.27 , viable cells C C pyruvate −1 −1 ) for ) ), or 3 2 - - - - - )

© 2015 Nature America, Inc. All rights reserved. extracts. The mixtures were vortexed and centrifuged to separate two layers. two separate to centrifuged and vortexed were mixtures The extracts. repeated. Chloroform (0.25 ml) and water (0.5 ml) were added to the combined wells. Solvents were transferred to fresh microfuge tubes and the extraction was were terminated by adding 0.25 ml methanol containing 1 N HCl to the culture Hepatocytes were incubated agonistswith the for 30 andmin 2 h. Incubations nM). (10 insulin LTB4and ethanol), nM) (30 (0.1% vehicle of presence the in 1- with incubated were buffer HPSB in Lipogenesis from forsectioned H&E staining. Immunohistochemistry. most of the macrophages in WAT tolerance glucose injection, first clodronatedepleted treatment the described, previouslyAs performed. were tests from d 10 After d. 3 every injections groups inhibitor and vehicle in were givenclodronate mice mg kg 100 (IP; treatment, of weeks 2 After group). ( regimen HFD the of 12 week at beginning weeks 3 for daily from fat) for 14 weeks and the Ltb4r1 inhibitor (50 mg kg Clodronate treatment. before the glucose-uptake or glucose-production assays. ml ng 100 of dose the at h 12 for hepatocytes or myocytes L6 to treated and treatment. PT hepatocytes, respectively. uptake assays or glucose production assays were performed in L6 glucose myocytes andtransfection, of h 18 After hepatocytes. primary or myocytes L6 into siRNA Ctrl or Jnk transfect to used was (Roche) reagent transfection siRNA (catalog number 6568) were purchased from Cell Signaling. The X-tremeGENE LTB4before the treatment. Jnk siRNA and Ctrl siRNAnumber 6233) (catalog h 0.5 for nM) (100 inhibitor Jnk the with treated were myocytes L6 Sciences. siRNA. Jnk and inhibitor Jnk plates were dissolved in 1N NaOH for protein estimation. the on Cells radioactivity. counting for vials scintillation to transferred were each tube, vortexed intermittently for 15 min, centrifuged and the supernatants ion-exchange resins, AG-501x8 resins (BioRad). 200 mg of resins were added to glucose released into the medium by separating radiolabeled glucose by mixed bed doi: 10.1038/nm.3800 PT was purchased from Calbiochem (catalog number 516560) 516560) number (catalog Calbiochem from purchased was PT 14 C palmitate: DAG and ceramide production. 8-week-old mice (C57BL6/J) were (C57BL6/J) mice 8-week-old kcal fed (60% HFD Liver tissue was fixed and in andembedded paraffin The Jnk inhibitor was purchased from EMD EMD from purchased was inhibitor Jnk The 6 4 . −1 ) or) followed PBS, by and third second 14 C-palmitate (0.2 mM, 0.5 0.5 mM, (0.2 C-palmitate −1 ) ) was orally gavaged n = 12 in each each in 12 = Hepatocytes µ Ci ml Ci −1 −1 )

60. 59. ments and outcome assessment. experi during allocation to blinded not were investigators The randomized. not were experiments The size. sample predetermine to used was method cal Data display variance. normal CA). Diego, (San 6.0 Prism GraphPad using correction Bonferroni the with variance of analysis one-way by determined was treatments various between means the as expressed are presented values The software. 1.42q ImageJ using performed were tion analyses. data and Statistical in the DAG and ceramide spots was determined. radioactivity and vapor.off iodine scraped bywere Spots detected wereplates methods published the per as respectively kinase acid and ceramide-1-phosphate by the reactions with DAG kinase and ceramide method from other lipids by thin layer chromatography (TLC) by following a published to determine incorporation into total lipids. DAG and ceramides were separated The lower organic layers were evaporatedcollected, and aliquots were counted 67. 66. 65. 64. 63. 62. 61.

e W. He, Quehenberger, O. Bektas, M., Jolly, P.S., Milstien, S. & Spiegel, S. A specific ceramide kinase assay kinase ceramide specific A S. Spiegel, and & Jolly,P.S.,S. M., Milstien, Bektas, ceramides of Determination Y.A. Hannun, & D.K. Perry, A., Bielawska, two- A G. Zwingelstein, & M. Babili, El O., Koul, G., Brichon, J., Bodennec, Y.S.Lee, P.Li, D.Y. Oh, M. Lu, Acad. Sci. USA Sci. Acad. muscle. in not but liver and fat in resistance insulin causes knockout plasma. to measure cellular levels of ceramide. of levels cellular measure to assay. kinase (2001). diglyceride the by diglycerides species. of diacylglycerol and separation ceramide simultaneous for procedure chromatography thin-layer dimensional resistance. insulin diet-induced acids. fatty 3 omega sensitizing effects. insulin-sensitizing and inflammatory resistance. (2010). insulin diet-induced prevent 6 5 et al. et and their identities were confirmed by the formation of phosphatidic et al. et t al. et J. Lipid Res. Lipid J. et al. et et al. et NCoR repression of LXRs restricts macrophage biosynthesis of insulin- of biosynthesis macrophage restricts LXRs of repression NCoR Inducible nitric oxide synthase deficiency in myeloid cells does not does cells myeloid in deficiency synthase oxide nitric Inducible Adipose-specific peroxisome proliferator-activated receptor gamma receptor proliferator-activated peroxisome Adipose-specific GPR120 is an omega-3 fatty acid receptor mediating potent anti- potent mediating receptor acid fatty omega-3 an is GPR120 Inflammation is necessary for long-term but not short-term high-fat short-term not but long-term for necessary is Inflammation

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