Laís Weissmann,1 Paula G.F. Quaresma,1 Andressa C. Santos,1 Alexandre H.B. de Matos,1

Vínicius D’Ávila Bittencourt Pascoal,1 Tamires M. Zanotto,1 Gisele Castro,1 Dioze Guadagnini,1

Joelcimar Martins da Silva,2 Licio A. Velloso,1 Jackson C. Bittencourt,2 Iscia Lopes-Cendes,1

Mario J.A. Saad,1 and Patricia O. Prada1,3

IKK« Is Key to Induction ofInsulin Resistance in theHypothalamus, and Its InhibitionReverses ObesityDiabetes 2014;63:3334–3345 | DOI: 10.2337/db13-1817

IKK epsilon (IKK«) is induced by the activation of nuclearfactor-kB (NF-kB). Whole-body IKK« knockout mice ona high-fat diet (HFD) were protected from insulin resis-tance and showed altered energy balance. We demon-strate that IKK« is expressed in neurons and isupregulated in the hypothalamus of obese mice, contrib-uting to insulin and leptin resistance. Blocking IKK« in thehypothalamus of obese mice with CAYMAN10576 or smallinterfering RNA decreased NF-kB activation in this tissue,relieving the inflammatory environment. Inhibition of IKK«activity, but not TBK1, reduced IRS-1Ser307 phosphoryla-tion and insulin and leptin resistance by an improvementof the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hy-pothalamus. These improvements were independent ofbody weight and food intake. Increased insulin and leptinaction/signaling in the hypothalamus may contribute toa decrease in adiposity and hypophagia and an enhance-ment of energy expenditure accompanied by lower NPYand increased POMC mRNA levels. Improvement of hy-pothalamic insulin action decreases fasting glycemia,glycemia after pyruvate injection, and PEPCK protein ex-pression in the liver of HFD-fed and db/db mice, suggest-ing a reduction in hepatic glucose production. We suggestthat IKK« may be a key inflammatory mediator in thehypothalamus of obese mice, and its hypothalamic in-hibition improves energy and glucose metabolism.

In the hypothalamus, insulin and leptin are potentanorexigens (1). Insulin activates the insulin receptor

(IR), leading to tyrosine phosphorylation of IR substrate1 (IRS-1) and 2 (IRS-2). Once activated, IR substrates bindto and activate the enzyme phosphatidylinositol 3-kinase(PI3K), increasing protein kinase B (Akt) phosphoryla-tion. These events decrease food intake (1–11). Leptinsignaling in the hypothalamus occurs through leptin re-ceptor, Janus kinase 2 (JAK2), and signal transducer andactivator of transcription 3 (STAT3) activation. In addi-tion, leptin may act through the JAK2/IRS/PI3K pathway(12). Both leptin and insulin increase the transcription ofproopiomelanocortin (POMC), an anorexigenic neuropep-tide (6,13–15), and inhibit the transcription of Agouti-related peptide and neuropeptide Y (NPY), which areorexigenic neuropeptides (8).

High-fat feeding affects insulin and leptin signaling,contributing to dysregulation of hypothalamic energyhomeostasis control (16). Activation of serine kinasesc-jun N-terminal kinase and inhibitor of kB kinase(IKKb) induces inhibitory IRS-1 serine phosphorylation,trigging insulin resistance (17,18). It is well known thatIKKb is activated in hypothalamic neurons of obese mice,inducing insulin and leptin resistance. Inhibition of hypo-thalamic IKKb improves insulin and leptin sensitivity,preventing diet-induced obesity (DIO) (18).

IKK epsilon (IKKe), also known as inducible IKK, ismainly expressed in immune cells, thymus, and spleen.Also a serine kinase, IKK is induced by the activation ofnuclear factor-kB (NF-kB) (19). That IKKe might regulatethe p65 NF-kB subunit and amplify inflammatory signals

1Department of Internal Medicine, State University of Campinas (UNICAMP),Campinas, São Paulo, Brazil2Department of Anatomy, Institute of Biomedical Sciences, University of SãoPaulo, São Paulo, Brazil3School of Applied Sciences, State University of Campinas (UNICAMP), Campinas,São Paulo, Brazil

Corresponding author: Patricia O. Prada, pprada@fcm.unicamp.br.

Received 28 November 2013 and accepted 28 April 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1817/-/DC1.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

3334 Diabetes Volume 63, October 2014

OBESITY

STUDIES

through the activation of other transcription factors is ofinterest (20–23).

IKKe levels and activity have been shown to be higherin the liver and adipose tissue of animals fed a high-fatdiet (HFD) (24). Whole-body IKKe knockout (KO) micewere protected from DIO and insulin resistance, suggest-ing an important role of IKKe in mediating insulin resis-tance induced by HFD (24). Of note, despite the leannesson an HFD, IKKe KO mice had higher food intake, VO2,body temperature, and uncoupling protein 1 (UCP-1) ex-pression in white adipose tissue, suggesting an alterationin the regulation of energy balance (24).

The enhanced IKKe expression in adipose tissue, liver,and adipose tissue macrophages of DIO mice, togetherwith the complex IKKe KO phenotype, indicate that thisserine kinase is key to the regulation of metabolism. How-ever, the regulation of IKKe in the hypothalamus and itsrole in the energy balance of DIO mice have not yet beeninvestigated. Thus, the aim of the current study was toinvestigate the expression and regulation of IKKe in thehypothalamus of obese mice and whether IKKe plays arole in regulating energy balance and hepatic glucose me-tabolism in the liver.

RESEARCH DESIGN AND METHODS

All experiments were approved by the Ethics Committee ofthe State University of Campinas. Eight-week-old maleC57BL/6J and db/db mice obtained from the State Univer-sity of Campinas, São Paulo, were assigned to receive a stan-dard rodent chow or an HFD with 55% calories from fat aspreviously described (25,26) and water ad libitum.

Intracerebroventricular CannulationAnesthetized mice underwent stereotaxic implantation(Model 963 Ultra Precise Small Animal Stereotaxic In-strument; Kopf) of 26-gauge stainless steel cannulas(Plastics One) in the right-side lateral ventricle as de-scribed previously (27).

IKK« InhibitionTo inhibit IKKe in the hypothalamus, we used the phar-macological inhibitor CAY10576 (Cayman Chemical Com-pany, Merck KGaA, Darmstadt, Germany) 60 mmol/Ldiluted in 10% DMSO in saline (vehicle). CAY or vehiclewere injected through intracerebroventricular (ICV) can-nulation in obese mice twice a day (0800 h and 1700 h)for 5 days. In another set of animals, we inhibited theexpression of IKKe by small interfering RNA (siRNA):RNA sense: 59 [Phos] rCrCrGrGrCrArGrArArGrGrUrGrCrUrArArUrCrArU 39 and RNA antisense: 59 [Phos]rGrArUrUrArGrCrArCrCrUrUrCrUrGrCrCrGrGrCrU 39 andits control green fluorescent protein (scramble): RNAsense: 59 [Phos] rCrArGrGrCrUrArCrUrUrGrGrArGrUrGrUrArUdTdT 39 and RNA antisense: 59 [Phos] rArUrArCrArCrUrCrCrArArGrUrArGrCrCrUrGdTdT 39, which wereinfused continuously by an ICV micro-osmotic pump(Alzet 1007D; DURECT Corporation, Cupertino, CA) dur-ing the 5 days.

Metabolic ParametersBody weight, fat mass, UCP-1 protein expression in adiposetissue, PEPCK protein expression in liver, and pyruvate weremeasured after 5 days of ICV treatments. Free-feedingmice were singly housed in a metabolic cage (3701M081;Tecniplast, Buguggiate, Varese, Italy) for acclimatization for5–7 days. Cumulative food intake was calculated by sum-ming the values of 24 h of food intake during the 5 days oftreatment. In another set of mice, 4 and 8 h of food intakewere recorded in response to ICV injection of humanrecombinant insulin 2 mg (Eli Lilly and Co., Indianapolis,IN) or leptin 10 ng (Calbiochem, San Diego, CA).

Pyruvate TestTwelve-hour–fasted mice were injected with sodium py-ruvate 2 g/kg i.p. Blood samples were collected from thetail immediately before and at 15, 30, 60, 90, 120, 150,and 180 min after the pyruvate injection.

VO2/VCO2 and Respiratory Exchange RatioDeterminationVO2, VCO2, and respiratory exchange ratio (RER) weremeasured in fed mice through an indirect open-circuitcalorimeter (Oxymax Deluxe System; Columbus Instru-ments, Columbus, OH) as described previously (27).Mice were allowed to adapt 2 days before. Measurementswere done on the last day of IKKe inhibition.

Proinflammatory Signals MeasurementsHypothalami and blood were collected after decapitationfrom fasted mice treated with CAY or vehicle. We deter-mined interleukin (IL)-1b, IL-6, and tumor necrosis factor-a(TNF-a) levels in serum and NF-kB in cellular nuclei oflysates obtained from hypothalami by using commerciallyavailable ELISA kits (Pierce Biotechnology Inc., Rockford,IL) following the manufacturer’s instructions.

ImmunofluorescenceImmunofluorescence was done as described before (28).We used anti-IKKe (D20G4-Rabbit mAb; Cell SignalingTechnology, Danvers, MA) 1:1,000 dilution, anti-neuronalnuclei (NeuN), a neuronal marker (MAB377-Mouse; MilliporeCorporation, Billerica, MA) 1:1,000 dilution, and anti-S100 b-antibody, an astrocyte marker (ab41548; Abcam).The negative controls were done by omitting the primaryantibodies.

ImmunoblottingHypothalami were removed and homogenized in buffer asdescribed previously (27). Phospho-IR, UCP-1, PEPCK,phospho-JAK2, phospho-STAT3, and phospho-IKKa/bantibodies were from Santa Cruz Technology (SantaCruz, CA). IKKe, phospho-Akt, JAK2, Akt, and TBK1were from Cell Signaling Technology.

IKK« and TBK1 Immunocomplex Kinase AssayHypothalami were collected from C57BL/6J mice fed chowor HFD treated with vehicle, CAY, or IKKe siRNA, homoge-nized with lysis buffer (50 mmol/L Tris [pH 7.5], 150mmol/L NaCl, 2 mmol/L EDTA, 5 mmol/L NaF, 25 mmol/L

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B-glycerophosphate, 1 mmol/L sodium orthovanadate,10% glycerol, 1% Triton X-100, 1 mmol/L dithiothreitol,and 1 mmol/L phenylmethylsulfonyl fluoride) in the pres-ence of protease inhibitors (Sigma-Aldrich), and immuno-precipitation was performed as described previously (19).The pellets containing purified kinases (IKKe or TBK1)were incubated in kinase buffer containing 25 mmol/LTris (pH 7.5), 10 mmol/L MgCl2, 1 mmol/L dithiothreitol,and 10 mmol/L ATP and in 1 mg myelin basic protein(MBP) (Millipore) per sample as a substrate for 30 minat 30°C. The kinase reaction was stopped by adding 43SDS buffer and warmed for 5 min at 95°C. Supernatantswere resolved by SDS-PAGE, transferred to nitrocellulose,and incubated with antiphosphoserine (ab1603; Abcam).The bands were analyzed by autoradiography and quanti-fied using UN-SCAN-IT software.

RNA Extraction and Real-Time PCRTwenty-four-hour–fasted mice treated for 5 days withCAY10576 or vehicle were injected ICV with saline or in-sulin. The hypothalami were harvested after 6 h, quicklyfrozen in liquid nitrogen, and stored at 280°C until RNAprocessing. Total RNA was obtained with an RNeasy MiniKit (Cat. no. 74106; Qiagen, Alameda, CA). QuantitativePCR was done using TaqMan PCR Master Mix (AppliedBiosystems) as previously described (25). Primer and probesequences were purchased from Applied Biosystems andwere NPY, Mm00445771_m1; POMC, Mm00435874_m1;and GAPDH, Mm00475829_g1 for mouse.

Statistical AnalysisResults are expressed as mean 6 SD. The significance wasdetermined by two-tailed Student t test, one- or two-wayANOVA with Bonferroni posttest, as appropriate, and dif-ferences were considered significant if P , 0.05. We usedGraphPad Prism software (GraphPad Software, San Diego,CA) for all statistical analyses.

RESULTS

IKK« Expression and Activity in the HypothalamusWere Increased in Obese MiceIKKe protein expression was detected in the hypothala-mus of control mice by immunoblotting, using the spleenas a positive control (Fig. 1A). Mice on an HFD and db/dbmice had increased protein levels of IKKe in the hypothal-amus compared with mice on a chow diet (Fig. 1B). Thesedata were supported by immunofluorescence that showedhigher staining for IKKe in the hypothalamus of mice onHFD compared with control mice (Fig. 1C). Using doublestaining for IKKe and NeuN markers or IKKe and astro-cytes (s100 b-marker), we showed that IKKe is expressedpredominantly in neurons (Fig. 1C), and there is no colo-calization of IKKe with astrocytes in the hypothalamus(Supplementary Fig. 1A). The negative controls for theimmunostaining for IKKe, NeuN, and s100 b-antibodiesare shown in the Supplementary Fig. 1B. The activity ofIKKe in the hypothalamus of mice on an HFD treated withvehicle was increased compared with mice on chow (Fig.

1D). This activity was blunted in the hypothalamus after 5days of CAY or IKKe siRNA treatment. TBK1, which is alsoa serine kinase inhibited by CAY, was not inhibited by IKKesiRNA treatment (Fig. 1D). IKKa/b phosphorylation wasnot altered by CAY or siRNA treatment (Fig. 1E).

Inhibition of Hypothalamic IKK« With CAY DecreasedAdiposity and Food Intake and Increased EnergyExpenditure and Thermogenesis in Mice Fed an HFDTo evaluate whether the inhibition of IKKe affected energyhomeostasis, we treated the mice on an HFD with ICV CAYfor 5 days. ICV CAY injections decreased the body weightand epididymal and retroperitoneal fat mass of mice fed anHFD compared with the vehicle group (Fig. 2A–C). ICVCAY treatment decreased cumulative food intake comparedwith vehicle treatment (Fig. 2D). Accordingly, mRNA levelsof the orexigenic neuropeptide NPY were lowered, andmRNA levels of the anorexigenic neuropeptide POMC wereelevated in the hypothalamus of fasted mice fed an HFDand treated with CAY compared with vehicle-treated miceon HFD (Fig. 2E and F). In addition, ICV CAY treatmentenhanced VO2 compared with mice treated with vehicle.VCO2 and RER were not different (Fig. 2G–I). Furthermore,we observed elevated UCP-1 protein levels in the brownadipose tissue of mice on an HFD that received CAY treat-ment compared with the vehicle group (Fig. 2J).

Inhibition of Hypothalamic IKK« With CAY ImprovedMetabolic Profiles and Reduced ProinflammatorySignals in Mice Fed an HFDLong-term treatment with ICV CAY decreased fastingblood glucose, insulin, and leptin levels (Fig. 3A–C). Toinvestigate whether the inhibition of IKKe interferedwith proinflammatory signals, we measured serum cyto-kine levels of obese mice treated with CAY or vehicle. Therewas no difference in the serum IL-1b and IL-6 levels ofCAY- and vehicle-treated mice. Serum TNF-a levels weredecreased in mice on an HFD treated with CAY (Fig. 3D–F).CAY-treated mice showed improved insulin sensitivitycompared with vehicle-treated mice during an insulin tol-erance test (Fig. 3G). To evaluate hepatic glucose produc-tion, we performed a pyruvate test, which indicated thatICV CAY treatment decreased glycemia after intraperito-neal pyruvate injection, suggesting lower hepatic glucoseproduction compared with vehicle-treated mice (Fig. 3H).This result was associated with lower PEPCK protein levelsin the liver of mice on HFD treated with CAY (Fig. 3I).

Inhibition of Hypothalamic IKK« With CAY ImprovedInsulin and Leptin Action and Signaling in theHypothalamus of Mice Fed an HFDAs expected, there was no change in food intake inresponse to insulin in mice on an HFD treated withvehicle. However, CAY treatment decreased food intakeafter 4, 8, and 24 h of insulin injection (Fig. 4A–C). Next,we investigated whether CAY treatment for 5 days af-fected insulin signaling in the hypothalamus of mice on anHFD. There was an improvement in IR and Akt phosphor-ylation and IRS-1/PI3K association in response to insulin in

3336 IKKe, Obesity, Insulin/Leptin, Hypothalamus Diabetes Volume 63, October 2014

the hypothalamus of obese mice treated with CAY com-pared with vehicle-treated mice (Fig. 4D–F). Accordingly,inhibition of IKKe with CAY decreased IRS-1Ser307 phos-phorylation in the hypothalamus (Fig. 4G). The p65 NF-kBlevels in the hypothalamus were decreased in mice treatedwith CAY compared with the vehicle group (Fig. 4H). Asexpected, there was no change in food intake in responseto leptin in mice on an HFD treated with vehicle. How-ever, CAY treatment decreased food intake after 4, 8, and24 h of leptin injection (Fig. 4I–K). These results wereaccompanied by increased JAK2 and STAT3 phosphoryla-tion, IRS-1/PI3K association, and IRS-2/PI3K associationin response to leptin in the hypothalamus of CAY-treatedmice (Fig. 4L–O).

Hypothalamic Insulin and Leptin Signaling WasImproved After CAY Treatment of Pair-Fed Mice

To exclude the interference of adiposity and food intakeon insulin and leptin signaling in the hypothalamusof mice treated with CAY, we performed pair-feeding

experiments. Thus, animals received the same amount ofan HFD during ICV treatment with vehicle or CAY (Fig.5A). Of note, CAY treatment induced a severe weight lossand a reduction of epididymal and retroperitoneal fatmass compared with the vehicle group, even thoughthey received the same amount of HFD (Fig. 5B–D). IRand Akt phosphorylation or JAK2 and STAT3 phosphor-ylation, in response to insulin and leptin, respectively, werehigher in the hypothalamus of mice treated with CAY thanin vehicle-treated mice (Fig. 5E–H). ICV CAY treatment in-creased VO2 consumption. VCO2 and RER were not differ-ent in mice treated with CAY or vehicle (Fig. 5I–K). Inaddition, we observed elevated UCP-1 protein levels in thebrown adipose tissue of mice on an HFD that received CAYtreatment (Fig. 5L).

Insulin and Leptin Signaling Was Improved After CAYTreatment of Pair-Weight MiceTo definitively exclude the interference of adiposity oninsulin and leptin signaling in the hypothalamus of mice

Figure 1—Expression and activity of IKKe in hypothalamus of mice. A: IKKe protein expression in spleen and hypothalamus of DIO mice. B:IKKe protein expression in hypothalamus from chow-fed, DIO, and db/db mice by immunoblotting. C: IKKe protein expression in hypo-thalamus from control (first two panels) and DIO mice by immunofluorescence using double staining for IKKe and NeuN markers. Whitearrows indicate the marked cells in red or green or with red and green. D: IKKe and TBK1 activity by in vitro kinase assays. E: IKKa/bphosphorylation in the hypothalamus after ICV treatment with CAY10576, siRNA, or vehicle in DIO mice. b-Actin was used as a loadcontrol. 3V, third ventricle; bv, blood vessel; Hyp, hypothalamus; IB, immunoblot; IP, immunoprecipitation; MBP-ser, myelin basic protein;VEH, vehicle; VMH, ventral medial hypothalamus.

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treated with CAY, we performed pair-weight experiments.Thus, animals received different amounts of HFD tomaintain the same body weight during CAY and vehicletreatments (Fig. 6A). At the end of the treatments, therewas no difference in epididymal and retroperitoneal fatmass between the two groups (Fig. 6B and C). In this con-text, vehicle-treated mice received less HFD than the CAYgroup (Fig. 6D). The results of insulin and leptin signalingwere similar to the results obtained in the pair-feedingexperiments. IR and Akt phosphorylation or JAK2 andSTAT3 phosphorylation in response to insulin or leptin, re-spectively, were higher in the hypothalamus of mice treatedwith CAY than in vehicle-treated mice (Fig. 6E–H).

Inhibition of Hypothalamic IKK« With siRNA ImprovedMetabolic Parameters in Mice Fed an HFDTo increase the specificity of IKKe inhibition in thehypothalamus, we used ICV injections by micro-osmoticpump with IKKe siRNA or scramble siRNA as a control for5 days. IKKe siRNA decreased IKKe mRNA levels in miceon an HFD (Fig. 7A). Accordingly, there was a decrease inhypothalamic IKKe protein expression in mice treated withsiRNA compared with the scramble group (Fig. 7B). Weight

loss was increased and epididymal and retroperitoneal fatmass decreased in siRNA-treated mice (Fig. 7C–E). Foodintake was lower and Vo2 higher in siRNA-treated micecompared with the scramble group (Fig. 7F and G). VCO2

was not different between the groups. In contrast, RER waslower in the siRNA group than in the scramble group (Fig.7H and I). UCP-1 expression was increased in the brownadipose tissue of siRNA-treated mice (Fig. 7J). IR phos-phorylation and Akt phosphorylation were enhanced inresponse to insulin in the hypothalamus of obese micetreated with siRNA. IRS-1 serine phosphorylation was de-creased after siRNA treatment (Fig. 7K–M). Fasting bloodglucose and PEPCK protein expression in liver were de-creased in the IKKe siRNA group (Fig. 7N and O).

Inhibition of Hypothalamic IKK« With CAY DecreasedBody Weight and Improved Energy and GlucoseMetabolism of db/db MiceIKKe expression was higher in the hypothalamus of db/dbmice (Fig. 1B). Thus, we used ICV CAY injections for5 days to inhibit IKKe activity in this animal model. Theactivity of IKKe in the hypothalamus of db/db mice waslower after CAY injections compared with vehicle-injected

Figure 2—Metabolic characteristics of mice on HFD treated with CAY10576. Body weight (A), epididymal fat mass (B), retroperitoneal fatmass (C), and cumulative food intake (5 days) (D) after ICV treatment with CAY or vehicle. NPY (E) and POMC (F ) mRNA levels in thehypothalamus of 24-h–fasted mice. VO2 (G) and VCO2 (H) in CAY-treated mice. RER (I) and UCP-1 (J) protein expression in brown adiposetissue in CAY- or vehicle-treated mice. Data are mean 6 SD from 5–10 mice. Two-tailed Student t test and two-way ANOVA were used.*P < 0.05 vs. vehicle. AU, arbitrary unit; BW, body weight; IB, immunoblot; VEH, vehicle.

3338 IKKe, Obesity, Insulin/Leptin, Hypothalamus Diabetes Volume 63, October 2014

mice (data not shown). CAY treatment induced a greaterweight loss and a reduction of epididymal and retroperi-toneal fat mass compared with the vehicle-treated db/dbmice (Fig. 8A–C). This result was accompanied by a reduc-tion in food intake and increased VO2. VCO2 was similar inthe two groups (Fig. 8D–F). RER was not different be-tween the groups (Fig. 8G). UCP-1 protein expressionwas elevated in brown adipose tissue of db/db mice treatedwith CAY (Fig. 8H). In addition, CAY treatment decreasedfood intake after 4 and 8 h of insulin injection, and IRphosphorylation and Akt phosphorylation were enhancedin response to insulin in the hypothalamus of db/db micetreated with CAY (Fig. 8I–L). Furthermore, we observeda decrease in fasting blood glucose and PEPCK proteinlevels in the liver (Fig. 8M and N).

DISCUSSION

In the current study, we showed that in the hypothalamusof DIO and genetic obese mice, there was an increase in

IKKe protein expression and activation. In addition, IKKeinhibition reduced adiposity and food intake, increasedenergy expenditure, and improved inflammation and glu-cose metabolism.

Obesity is associated with low-grade inflammation invarious tissues, including the hypothalamus (17,28,29).Activation of the IKK/NF-kB pathway is a marker of in-flammatory signals in the hypothalamus (18). It wasshown before that hypothalamic neuronal IKKb playsan important role in controlling energy homeostasis(18). In the current study, we demonstrate that IKKe ispredominantly expressed in neurons not in astrocytes andmay affect the neuronal regulation of energy balance.

The results showed that the inhibition of hypothalamicIKKe reduced NF-kB activity in the hypothalamus, sug-gesting alleviation of the inflammatory environment inthis tissue. The molecular link between inflammationand insulin resistance remains to be completely estab-lished, although it is known that the NF-kB pathway plays

Figure 3—Metabolic profile and proinflammatory signals in mice fed an HFD after inhibition of hypothalamic IKKe with CAY. Fasting bloodglucose (A), serum insulin (B), leptin (C), IL-1b (D), IL-6 (E), and TNF-a (F) levels of mice treated with ICV vehicle or CAY. G: Blood glucoselevels during insulin tolerance testing in mice treated with ICV vehicle or CAY. H: Percent of initial blood glucose after injection of sodiumpyruvate 2 g/kg i.p. I: PEPCK protein expression in the liver of mice treated with CAY. Data are mean 6 SD from 5–10 mice. Two-tailedStudent t test and two-way ANOVA were used. *P < 0.05 vs. vehicle. IB, immunoblot; VEH, vehicle.

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Figure 4—Assessment of insulin and leptin action and signaling in mice on HFD treated with CAY. Food intake at 4 (A), 8 (B), and 24 h (C )after ICV injection of insulin in mice treated with CAY. D: IR phosphorylation. E: IRS-1 and PI3K association. Akt (F) and IRS-1Ser307 (G)phosphorylation. H: NF-kB levels in nuclear lysates. Food intake 4 (I), 8 (J), and 24 h (K) after ICV injection of leptin in mice treated with CAY.L: JAK2 phosphorylation. M: IRS-1 and PI3K association. N: IRS-2 and PI3K association. O: STAT3 phosphorylation. Data are mean 6 SDfrom 8–10 mice. Two-tailed Student t test (G and H) or one-way ANOVA with Bonferroni posttest was used. *P< 0.05 vs. vehicle. BW, bodyweight; IB, immunoblot; IP, immunoprecipitation; VEH, vehicle.

3340 IKKe, Obesity, Insulin/Leptin, Hypothalamus Diabetes Volume 63, October 2014

an important role (30). Disruption of the NF-kB pathwayby deletion of the canonical IKKb gene or by pharmaco-logical inhibition reverses insulin resistance in obese mod-els (18). However, the role of the noncanonical IKKe ispoorly understood.

Insulin and leptin resistance occurs before weight gainin rodents on an HFD, suggesting that abnormalities inthese hormones are likely to trigger the development ofobesity rather than the opposite (31). In the currentstudy, we observed impairment of insulin and leptin ac-tion and signaling in the hypothalamus of mice on anHFD. High-fat feeding promotes an inflammatory envi-ronment, leading to activation of serine kinases, inducingIRS-1 serine phosphorylation. This event triggers hypo-thalamic insulin resistance. IKKe blockage with CAY orsiRNA blunted IRS-1Ser307 phosphorylation without alter-ing the phosphorylation of IKKa/b. This observation

reinforces that hypothalamic IKKe is important in blunt-ing insulin signaling. Together with a reduction in IRS-1serine phosphorylation in the hypothalamus of CAY- andsiRNA-treated mice, we observed an enhanced IRS-1/PI3Kassociation in response to insulin, indicating an improve-ment of insulin resistance in this tissue. In addition, weshowed that CAY or siRNA treatment was able to reverseleptin resistance. This effect may be explained, at least inpart, by the reduction of IRS-1 serine phosphorylation.

The inhibitor CAY blocks another serine kinase, TBK1,in peripheral tissues (19). TBK1 and IKKe have been pro-posed as key regulators of metabolic function in mice(19). Their inhibition with the drug amlexanox was ableto improve metabolic dysfunction in obese mice (19). Inour experiments, we observed similar effects on energymetabolism and insulin signaling in the hypothalamus byusing CAY or siRNA. We argue that IKKe is the major

Figure 5—Hypothalamic insulin and leptin signaling after CAY treatment of pair-fed mice. Food intake (A), weight loss (B), epididymal (C),and retroperitoneal fat mass (D) in mice treated with CAY or vehicle. Hypothalamic IR (E) and Akt (F ) phosphorylation in response to ICVinsulin. Hypothalamic JAK2 (G) and STAT3 (H) phosphorylation in response to ICV leptin in mice treated with CAY or vehicle. VO2 (I), VCO2(J), and RER (K) in mice treated with CAY or vehicle. L: UCP-1 protein expression in brown adipose tissue of mice treated with CAY orvehicle. Data are mean6 SD from 8–10 mice. Two-tailed Student t test and one-way ANOVA with Bonferroni posttest were used. *P< 0.05vs. vehicle. BW, body weight; IB, immunoblot; VEH, vehicle.

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player impairing energy and glucose metabolism in obesemice because siRNA treatment is very specific, inhibitingIKKe but not TBK1, and we observed enhanced TBK1activity in the hypothalamus of siRNA-treated mice. Theincreased TBK1 activity may be due to direct activation bythe canonical IKKs IKKa and IKKb in response to inflam-matory inputs (32). In fact, TBK1 phosphorylation andactivation is increased in peritoneal macrophages fromIKKe KO mice, suggesting that in the absence of IKKe,TBK1 may be upregulated (19). In the current study, it isunknown which hypothalamic neuron displayed enhancedTBK1 activity because we used the whole hypothalamussamples in our experiments.

CAY treatment was associated with hypophagia inresponse to insulin and leptin, suggesting a reversion ofinsulin and leptin resistance in the hypothalamus. Theseeffects persisted even in pair-feeding and pair-weightexperiments, suggesting that the inhibition of IKKe andnot differences in body weight or food intake accountedfor the improvement in energy metabolism. The reductionin food intake may also be a reflection of improved insulinand leptin action and signaling. Indeed, we observed de-creased NPY and increased POMC gene expression, whichmay contribute to hypophagia. Chiang et al. (24) showedthat IKKe KO mice on chow or HFD had elevated foodintake. The difference in food intake from IKKe KO andinhibition of IKKe with CAY or siRNA may be due to thedifferent timing of IKKe inhibition. IKKe KO mice had

the IKKe gene deleted early in life and might have in-creased food intake to compensate for the elevated energyexpenditure.

Despite elevated food intake, IKKe KO mice were lean.The leanness was mainly due to enhanced energy expen-diture and thermogenesis (24). In the current study, weobserved that CAY or siRNA was associated with in-creased energy expenditure and thermogenesis, whichmay have been caused by improved leptin signaling andaction in the hypothalamus. We emphasize that the in-hibition of IKKe increased energy expenditure and wasprobably an important contributor to the leanness. Inthe pair-feeding experiments, control animals ingestedthe same amount of food as mice treated with CAY. Nev-ertheless, CAY-treated mice had lower adiposity than thevehicle group. This phenomenon suggests that IKKe isimportant in the regulation of energy expenditure anddeserves further investigation.

TNF-a, IL-6, and IL-1b are major enhancers of theinflammatory response (33). Evidence has demonstratedthat these cytokines play a role in regulating energy me-tabolism. The present data show a reduction in serumTNF-a levels, suggesting a decrease in inflammatory sig-nals. Decreased serum TNF-a levels might also have beendue to the reduction of fat mass observed in the CAY-treated mice.

Knockdown of IKKe in the whole body decreased fast-ing blood glucose levels and decreased blood glucose in

Figure 6—Hypothalamic insulin and leptin signaling after CAY treatment of pair-weight mice on HFD. Body weight (A), epididymal (B), andretroperitoneal (C) fat mass. D: Food intake of mice treated with CAY or vehicle. Hypothalamic IR (E ) and Akt (F) phosphorylation inresponse to ICV insulin. Hypothalamic JAK2 (G) and STAT3 (H) phosphorylation in response to ICV leptin in mice treated with CAY orvehicle. Data are mean 6 SD from 8–10 mice. Two-tailed Student t test or one-way ANOVA with Bonferroni posttest was used. *P < 0.05vs. vehicle. BW, body weight; IB, immunoblot; VEH, vehicle.

3342 IKKe, Obesity, Insulin/Leptin, Hypothalamus Diabetes Volume 63, October 2014

response to pyruvate (24,34). In contrast, overexpres-sion of IKKe in hepatoma cells enhanced expression ofselected gluconeogenic genes, suggesting direct and cell-autonomous effects in the liver. The present data showa similar result of inhibiting IKKe in the hypothalamusof HFD and db/db mice. This result on blood glucose was

accompanied by lower PEPCK expression in the liver,suggesting a reduction of hepatic glucose output. Studieshave shown that the action of insulin and leptin in thehypothalamus affects hepatic glucose production (35–37).Because db/db mice are leptin insensitive, we suggest thatan improvement of insulin action in the hypothalamus

Figure 7—The inhibition of hypothalamic IKKe with siRNA improved metabolic parameters in mice on HFD. A: IKKe mRNA levels in thehypothalamus of IKKe siRNA- or scramble (SCR)-treated mice. B: Protein levels of hypothalamic IKKe in mice treated with siRNA comparedwith SCR. Percent weight loss (C), epididymal fat mass (D), and retroperitoneal fat mass (E) of mice treated with IKKe siRNA or SCR. Foodintake (F), VO2 (G), VCO2 (H), and RER (I) in IKKe siRNA-treated mice. J: UCP-1 protein expression in brown adipose tissue of mice treatedwith IKKe siRNA or SCR. Hypothalamic IR (K) and Akt phosphorylation in response to ICV insulin (L) and IRS-1Ser307 phosphorylation (M) inmice treated with IKKe siRNA or SCR. Fasting blood glucose levels (N) and PEPCK protein expression (O) in liver of mice treated with IKKesiRNA or SCR. Data are mean6 SD from 8–10 mice. Two-tailed Student t test or one-way ANOVA with Bonferroni posttest was used. *P<0.05 vs. SCR. AU, arbitrary unit; BW, body weight; IB, immunoblot.

diabetes.diabetesjournals.org Weissmann and Associates 3343

induced by inhibition of IKKe might have been responsiblefor those phenotypes.

In summary, the data provide evidence that IKKe isexpressed in neurons and upregulated in the hypothalamusof obese mice and contributes to insulin and leptin resis-tance. Blocking IKKe in the hypothalamus of obese mice, atleast for a short period, decreases NF-kB activation, reduc-ing IRS-1 serine phosphorylation in the hypothalamus. In-hibition of IKKe activity but not TBK1 decreases insulinand leptin resistance by improving the IR/IRS-1/Akt andJAK2/STAT3 pathways in the hypothalamus of obese mice.These improvements were independent of body weight and

food intake. Increased insulin and leptin action and signal-ing in the hypothalamus may contribute to the reductionin adiposity and hypophagia and enhancement of energyexpenditure accompanied by lower NPY and increasedPOMC mRNA levels. Improvement of insulin actiondecreases fasting glycemia, glycemia after pyruvate injec-tion, and PEPCK protein expression in the liver, suggest-ing a reduction in hepatic glucose production. The resultsof this study suggest that the noncanonical IkB kinaseIKKe may be a key inflammatory mediator in the hypo-thalamus of obese mice, and its hypothalamic inhibitionmeliorates energy and glucose metabolism.

Figure 8—The inhibition of hypothalamic IKKe with CAY in db/dbmice. Percent weight loss (A), epididymal fat mass (B), and retroperitonealfat mass (C) of db/dbmice treated with CAY. D: Cumulative food intake (5 days) during treatment with CAY or vehicle. VO2 (E), VCO2 (F ), andRER (G). H: UCP-1 protein expression in brown adipose tissue of db/db mice treated with CAY or vehicle. Food intake 4 (I ) and 8 h (J) afterICV injection of insulin in mice treated with CAY. IR (K) and Akt phosphorylation (L) in response to ICV insulin. Fasting blood glucose levels(M) and PEPCK protein expression (N ) in liver of db/db mice treated with CAY. Data are mean 6 SD from 4–6 mice. Two-tailed Studentt test was used. *P < 0.05 vs. vehicle. BW, body weight; IB, immunoblot; VEH, vehicle.

3344 IKKe, Obesity, Insulin/Leptin, Hypothalamus Diabetes Volume 63, October 2014

Acknowledgments. The authors thank L. Janeri and J. Pinheiro (Depart-ment of Internal Medicine, UNICAMP, Campinas, São Paulo, Brazil) for technicalassistance.Funding. This work was supported by FAPESP (Fundação de Amparo à Pesquisado Estado de São Paulo): 2012/10338-6 and 2010/52068-0, CEPID (Centros dePesquisa, Inovação e Difusão): 2013/07607-8, São Paulo, Brazil, and CNPq INCT(Instituto Nacional de Ciência e Tecnologia de Obesidade e Diabetes): 573856/2008-7 and UNIVERSAL: 481084/2013-4.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.W. and P.O.P. contributed to researchingdata and to the design and performance of the experiments, data analysis,discussion, and writing and review of the manuscript. P.G.F.Q., A.C.S., A.H.B.d.M.,V.D.B.P., T.M.Z., G.C., D.G., J.M.d.S., and J.C.B. contributed to the performanceof the experiments and data analysis. L.A.V., J.C.B., I.L.-C., and M.J.A.S. con-tributed to the discussion and writing and review of the manuscript. P.O.P. is theguarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of the dataanalysis.

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