Biochimica et Biophysica Acta 1852 (2015) 1049–1058

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Biochimica et Biophysica Acta

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PPARβ/δ ameliorates fructose-induced insulin resistance in adipocytes bypreventing Nrf2 activation

Emma Barroso a,b, Rosalía Rodríguez-Rodríguez a, Matilde R. Chacón b,c,d, ElsaMaymó-Masip b,c,d, Laura Ferrer a,Laia Salvadó a,b, Emilio Salmerón a, Martin Wabistch e, Xavier Palomer a,b, Joan Vendrell b,c,d,Walter Wahli f,g, Manuel Vázquez-Carrera a,b,⁎a Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry and Institut de Biomedicina de la UB (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spainb CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)–Instituto de Salud Carlos III, Barcelona, Spainc Endocrinology and Diabetes Unit, Research Department, University Hospital of Tarragona Joan XXIII, Pere Virgili Institute, Tarragona, SpaindRovira i Virgili University, Tarragona, Spain

e Divisions of Paediatric Endocrinology and Diabetes, Ulm University, Ulm, Germanyf Center for Integrative Genomics, National Research Center Frontiers in Genetics, University of Lausanne, Quartier UNIL-Sorge, Bâtiment Génopode, CH-1015 Lausanne, Switzerlandg Lee Kong Chian School of Medicine, Nanyang Technological University, The Academia, 20 College Road, 169856, Singapore

⁎ Corresponding author at: Manuel Vázquez-Carrera, Ude Farmàcia, Diagonal 643, E-08028 Barcelona, Spain. Te93 403 5982.

E-mail address: mvazquezcarrera@ub.edu (M. Vázque

http://dx.doi.org/10.1016/j.bbadis.2015.02.0100925-4439/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 December 2014Received in revised form 13 February 2015Accepted 21 February 2015Available online 26 February 2015

Keywords:PPARβ/δAdipocyteFructoseCD36JNKOxidized LDL

Westudiedwhether PPARβ/δdeficiencymodifies the effects of high fructose intake (30% fructose in drinkingwater)on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhancesadipose tissue inflammation and impairs insulin signaling. Fructose intake for 8 weeks significantly increasedbody and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-typemice. Feeding PPARβ/δ-deficientmicewith fructose exacerbated glucose intolerance and led tomacrophage infiltra-tion, inflammation, enhancedmRNA and protein levels of CD36, and activation of the JNK pathway inwhite adiposetissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhib-ited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interest-ingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubationwithGW501516 abolished this increase. In agreementwithNrf2 playing a role in the fructose-induced CD36proteinlevel increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulatedAKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target geneNAD(P)H:quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting thatPPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficientmice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

The drastic increase in the incidence of obesity and type 2 diabetesmellitus observed in Western countries over recent decades has coin-cided with an increase in total energy consumption and a shift in thetypes of nutrients ingested. This includes a dramatic increase in theconsumption of fructose, used in processed or prepared foods andcaloric beverages [1]. Strong evidence suggests that diets high in fruc-tose result in hepatic steatosis, hyperlipidemia and insulin resistance[2,3]. Unlike glucose, which is widely used by tissues throughout the

nitat de Farmacologia, Facultatl.: +34 93 402 4531; fax: +34

z-Carrera).

body, fructose is mainly metabolized in the liver, where it increases denovo lipogenesis [4]. As a result, the effects of fructose overconsumptionhave mainly been studied in liver. However, high fructose intake is alsoassociated with adiposity [2,3]. In fact, in a recent study conducted inadolescents, greater fructose consumptionwas associatedwithmultiplemarkers known to increase the risk of cardiovascular disease and type 2diabetes mellitus, and it appeared that these relationships weredependent on visceral adiposity [5].

The nuclear receptor peroxisome proliferator-activated receptor(PPAR)β/δ is an important regulator of lipid and energy metabolism inadipose tissue and skeletal muscle [6,7]. Therefore, PPARβ/δ representsa candidate mediator of insulin resistance and type 2 diabetes mellitus,and a promising target for the prevention and treatment of these dis-eases [6,8]. Thus, a reduction in PPARβ/δ activity can contribute toinflammation and insulin resistance. For instance, genetic variation in

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this gene predicts the conversion from impaired glucose tolerance totype 2 diabetes mellitus [9]. In addition, we recently reported that inseverely obese insulin-resistant patients, enhanced inflammation invisceral adipose tissue was accompanied by an increase in PPARβ/δmRNA levels. This increase in the expression of PPARβ/δ seems to be acompensatory mechanism since PPARβ/δ activity was reduced inhuman cultured adipocytes exposed to inflammatory stimuli [10].Overall, these findings suggest the hypothesis that PPARβ/δ plays acentral role in metabolic regulation and its deficiency might contributeto exacerbating metabolic derangements caused by fructose.

The aim of thisworkwas to studywhether PPARβ/δ deficiencymod-ifies the effects of a high fructose intake (30% fructose solution as theonly fluid source) on glucose tolerance and adipose tissue dysfunction.We focused on the CD36-dependent pathway that enhances adiposetissue inflammation and impairs insulin signaling [11]. This class Bscavenger receptor recognizes a variety of ligands, including oxidizedLDL (oxLDL), which contribute to the pathogenesis of insulin resistance.In fact, CD36 is highly expressed in adipocyteswhere it drives an inflam-matory processwhich involves the activation of c-JunN-terminal kinase(JNK) and the consequent disruption of insulin signaling [11]. Thesechanges are dependent on CD36 since its absence protects mice frominsulin resistance associated with diet-induced obesity [11].

Interestingly, activation of the nuclear factor E2-related factor 2(Nrf2), one of themost regulated defensemechanisms against oxidativestress, upregulates Cd36 expression [12] and impairs insulin signalingand glucose uptake [13]. Under physiological conditions, Nrf2 binds toKelch-like ECH associating protein (Keap 1) in the cytoplasm. On expo-sure to oxidative stress, Nrf2 releases from Keap 1, translocates to thenucleus and binds to antioxidant-responsive elements (ARE) locatedin the promoter of its target genes, including Cd36 [12].

Here we report that the combination of a high fructose intake andPPARβ/δ deficiency increases CD36 protein levels via Nrf2, a processthat promotes inflammation and glucose intolerance.

2. Methods

2.1. Materials

GW501516 was purchased from Alexis Biochemicals (Lausen,Switzerland). All other chemicals, except where specified, were fromSigma-Aldrich (St. Louis, MO).

2.2. Animals and treatments

Mice were maintained under standard conditions of illumination(12-h light/dark cycle) and temperature (21 ± 1 °C). Food and waterwere provided ad libitum. All procedures were conducted in accordancewith the principles and guidelines established by the University ofBarcelona Bioethics Committee, as stated in Law 5/21 July 1995 passedby the Generalitat de Catalunya and the Commission de Surveillancede l'Expérimentation Animale of the Canton of Vaud (Switzerland).

Four- to five-month-old male PPARβ/δ knockout mice and controlmice (PPARβ/δ+/+, wild-type) with the same genetic background(C57BL/6X129/SV) and an initial weight of 20–25 g were fed a standarddiet. The generation of PPARβ/δ nullmicehas been described previously[14]. In addition to normal chow, the mice were fed either a 30%fructose solution or plain tap water for 12 weeks, as previously de-scribed [15,16].

Before the end of the treatments, a glucose tolerance test was per-formed on mice after fasting for 4 h. Animals received 2 g/kg bodyweight of glucose by intraperitoneal injection and blood was collectedfrom the tail vein after 0, 15, 30, 60 and 120 min. The body weight ofthe mice was measured regularly during the treatment period. At theend of the treatments, the mice were killed under isoflurane anesthesiaand samples of liver and epididymalwhite adipose tissuewere collectedand frozen in liquid nitrogen, and then kept at−80 °C.

2.3. Measurement of malondialdehyde in white adipose tissue samples

The content of malondialdehyde (MDA), a marker of lipid peroxida-tion, was determined using the lipid peroxidation (MDA) assay kit(Sigma) in adipose tissue.

2.4. Cell culture

The human Simpson–Golabi–Behmel Syndrome (SGBS) cell line ofpreadipocytes was induced to differentiate to mature adipocytes asdescribed previously [17]. During the differentiation process glucosein the medium was reduced from 17 to 5 mmol/l and adipocytes wereexposed to either 25 mmol/l fructose or mannitol as a control, in orderto rule out the possibility that the effects of fructose were not the resultof an increase in the osmotic pressure, in the presence or absence ofeither 10 μmol/l GW501516 (PPARβ/δ agonist) or 10 μmol/l GSK0660(PPARβ/δ antagonist).

3T3-L1 preadipocytes (ATCC)were grown to confluence in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% bovine calfserum. Two days after confluence (day 0), differentiation of the 3T3-L1cells was induced in DMEM containing 10% fetal bovine serum (FBS),methylisobutylxanthine (500 μM), dexamethasone (0.25 μM), and insu-lin (5 μg/ml) for 48 h. The cells were then incubated in 10% FBS/DMEMwith insulin for three days and this was then replaced with FBS/DMEM.The medium was changed every 2 days. Fat droplets were observed inmore than 90% of the cells after day 10. The adipocytes were thenexposed to 25 mmol/l fructose or mannitol as a control in the presenceor absence of 10 μmol/l GW501516 or 0.5 μmol/l trigonelline. After incu-bation, RNA, whole cell lysates, and both cytosolic and nuclear proteinextracts were extracted from the cells as described below.

2.5. RNA preparation and quantitative RT-PCR

Relative levels of specific mRNAs were assessed by real-time reversetranscription-polymerase chain reaction (RT-PCR), as previously de-scribed [18]. Total RNAwas isolated using the Ultraspec reagent (Biotecx,Houston, TX). RNA sampleswere cleaned (NucleoSpin RNA II; Macherey-Nagel, Düren, Germany) and checked for integrity by agarose gel electro-phoresis. The total RNA isolated using this methodwas not degraded andfree from protein and DNA contamination. Reverse transcription wasperformed from 0.5 μg total RNA using Oligo(dT)23 and M-MLV ReverseTranscriptase (Invitrogen, San Diego, CA). The PCR reaction contained10 ng of reverse-transcribed RNA, 2× IQTM SYBRGreen Supermix(BioRad, Barcelona, Spain) and 900 nM of each primer. The PCR assayswere performed on a MiniOpticonTM Real-Time PCR system (BioRad).Thermal cycling conditions were as follows: activation of Taq DNApolymerase at 95 °C for 10 min, followed by 40 cycles of amplificationat 95 °C for 15 s and at 60 °C for 1min. Primer Express Software (AppliedBiosystems, Foster City, CA)was used to design the primers (Supplemen-tal Table S1). Optimal primer amplification efficiency for each primer setwas assessed and a dissociation protocol was carried out to ensure asingle PCR product. The results for the expression of specific mRNAs arealways presented relative to the expression of the control gene.

2.6. Isolation of nuclear extracts

Nuclear extracts were isolated was performed as describedelsewhere [10].

2.7. Histological studies

White adipose tissue was fixed in paraformaldehyde and embeddedin paraffin blocks using conventional histological techniques. Paraffinsections were cut at 10 μm, deparaffinized in xylene and rehydrated towater by ethanol at different percentages (100, 96, 80, 70 and 50).After hematoxylin–eosin staining of the tissue preparations, images

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were acquired with a BX41 (Olympus, Germany) microscope at 10×magnification. The cell area of 260–550 adipocytes per treatmentgroup was measured with the image analysis program ImageJ 1.42(Bethesda, MD) to determine the average cell size and size distributionfor each experimental group.

For immunohistochemistry, paraffin sections were deparaffinizedand rehydrated as described above. Slides were blocked and perme-abilized with PBS containing 1% bovine serum albumin (BSA) and 0.1%Triton X-100 for 20 min. After two 5-min washes in PBS, the slideswere incubated with the primary antibody for Cd11b (Millipore,Bedford, MA) overnight at 4 °C. Theywere thenwashed again and incu-bated for 1 h at room temperature in the dark with Alexa Fluor second-ary antibody. After washing again, nuclear staining was performed byincubating the slides in Hoechst (H-33258, Fluka, Madrid, Spain) at2 μg/ml in PBS for 10 min at room temperature in the dark. Finally, theslides were washed, mounted using Prolong Gold (Invitrogen) anti-fademedium, allowed to dry overnight at room temperature and storedat 4 °C. Images were acquired with an Olympus BX61 epifluorescencemicroscope.

2.8. Immunoblotting

To obtain total proteins, adipose tissue and adipocytes were homog-enized in RIPA lysis buffer with 5mMNaF, 1 mM phenylmethylsulfonylfluoride, 10 mM sodium orthovanadate and 5.4 μg/ml aprotinin at 4 °Cfor 30 min. The homogenate was centrifuged at 17,000 g for 30 min at4 °C. Protein concentration was measured by the Bradford method.Total proteins (30 μg) were separated by SDS-PAGE on 10% separationgels and transferred to Immobilon polyvinylidene difluoridemembranes(Millipore). Western blot analysis was performed using antibodiesagainst total and phospho-AKT (Ser473), total and phospho-JNK(Thr183/Tyr185), phospho-c-JUN (Ser73) (Cell Signaling TechnologyInc., Danvers, MA), total c-JUN, LOX-1, Nrf2, NQO1 (Santa CruzBiotechnology, Santa Cruz, CA), CD36 (Novus Biologicals, Littleton,CO), and β-actin (Sigma-Aldrich). Detection was achieved using theEZ-ECL chemiluminescence detection kit (Biological Industries IsraelBeit-Haemek Ltd., Kibbutz Beit-Haemek, Israel). The equality of theprotein loading was assessed using Ponceau S staining. The size of theproteins detected was estimated using protein molecular-massstandards (Life Technologies, Grand Island, NY).

2.9. Oxidized LDL

Serum levels of oxLDL were analyzed by ELISA (USCN Life Science,Houston, TX).

2.10. Statistical analysis

Results are expressed as mean ± S.D. of four to six separateexperiments. Significant differences were established by one-wayANOVA, using the GraphPad Instat program (GraphPad SoftwareV2.03) (GraphPad Software Inc., San Diego, CA). When significant dif-ferences were found, the Tukey–Kramer multiple comparisons post-test was applied. Differences were considered significant at p b 0.05.

3. Results

3.1. Fructose exacerbates glucose intolerance in PPARβ/δ-deficient mice

Water-fed PPARβ/δ-deficient mice showed a lower bodyweight andliver weight than wild-type mice, while fructose intake significantlyincreased both parameters in the PPARβ/δ-deficient mice only (Fig. 1Aand B). As expected, the increase in both parameters was compensatedfor in the liver weight–body weight ratio (Supplementary Fig. 1).Surprisingly, fructose feeding did not significantly increase liver triglyc-eride accumulation in wild-type mice, suggesting that the genetic

background of these mice might be resistant to hepatic steatosis. How-ever, this protective effect observed in fructose-fed wild-type mice dis-appeared in fructose-fed PPARβ/δ-deficient mice, since carbohydrateintake resulted in a marked accumulation of triglycerides in the liver(~2-fold increase, p b 0.05) compared to water-fed PPARβ/δ-deficientmice (Fig. 1C). When subjected to a glucose-tolerance test, whichevaluates the capacity of the body to adjust glucose levels after anacute glucose injection, water-fed PPARβ/δ-deficient mice wereglucose-intolerant compared to wild-type mice, as demonstratedby the significant increase in the area under the curve (AUC)(Fig. 1D and E). Interestingly, fructose intake significantly exacerbatedthis increase (p b 0.05 vs.water-fed PPARβ/δ-deficientmice), indicatingthat fructose potentiates glucose intolerance in these mice.

3.2. Fructose-induced adipocyte hypertrophy is exacerbated inPPARβ/δ-deficient mice

It has been well established that the larger the adipocyte, the lesssensitive it is to insulin [19]. When we examined the effects of fructoseon cell size in vivo we observed that adipocytes from water-fed wild-type mice were larger (63% adipocytes b 2000 μm2) than those ofwater-fed PPARβ/δ-deficient mice (87% adipocytes b 2000 μm2)(Fig. 2A and B), which is consistent with a previous study reportingthat PPARβ/δ-null mice contain smaller adipocytes than wild-type mice[20]. It is worth noting that fructose feeding led to an increase in thesize of the adipocytes in wild-type mice (21% adipocytes b 2000 μm2)and that this increase was even higher in PPARβ/δ-deficient mice (9.5%adipocytes b 2000 μm2). These findings indicate that, despite the lackof induction of triglyceride accumulation in the liver of wild-type mice,fructose led to adipocyte hypertrophy in these mice and this increasewas exacerbated in PPARβ/δ-deficient mice.

3.3. Increased pro-inflammatory cytokine and chemokine expressionand macrophage infiltration in white adipose tissue from fructose-fedPPARβ/δ-deficient mice

Adipocyte size is an important determinant of the expression andsecretion of pro-inflammatory adipokines, which is greater in largeradipocytes than in small or medium-sized cells [21,22]. Thus, we nextevaluated whether the changes in the size of the adipocytes were ac-companied by changes in the expression of pro-inflammatory cytokinesand chemokines. The expression of Tnf-α was significantly increasedin the white adipose tissue of fructose-fed PPARβ/δ-deficient mice(Fig. 3A). Since increased macrophage infiltration in adipose tissueis an important component of the chronic inflammatory process in-volved in the development of insulin resistance [23] and monocytechemoattractant protein (MCP-1) is a key chemokine involved in the re-cruitment of macrophages, we measured its expression. Mcp-1 mRNAlevels were strongly enhanced (p b 0.05) (Fig. 3B) in the white adiposetissue of fructose-fed PPARβ/δ-deficient mice. In agreement withthis, the expression of the macrophage markers F4/80 and Cd68 wassignificantly increased in white adipose tissue from fructose-fedPPARβ/δ-deficient mice (Fig. 3C and D). To confirm the increasedmacrophage content within adipose tissue, we performed immunohis-tochemistry with a Cd11b antibody. Only in white adipose tissue fromfructose-fed PPARβ/δ-deficient mice did we observe Cd11b-positivecrown-like structures, hallmarks of adipose tissue inflammation(Fig. 3E).

An important feature of adipose tissue inflammation and insulinresistance is the predominance of a classic M1 pro-inflammatory mac-rophage phenotype,which is amajor source of inflammatorymediators,such as TNF-α and MCP-1, vs. an M2 anti-inflammatory adipose tissue-resident macrophage. Interestingly, it has been reported that PPARβ/δregulates adipose tissue-resident macrophages thus leading towardthe anti-inflammatory M2 phenotype [20]. When we analyzed the ex-pression of two M2 macrophage markers, interleukin 10 (Il-10) and

Fig. 1. Fructose exacerbates glucose intolerance in PPARβ/δ-deficient mice. Wild-type (WT) and PPARβ/δ-deficient mice (PPARβ/δ−/−) were fed with either water or water containing30% fructose for eight weeks. A, body weight. B, liver weight. C, hepatic triglyceride content. D, glucose tolerance test. E, area under the curve (AUC). Data are expressed as mean ±S.D. (6 mice per group). ap b 0.05 vs.water-fed WT mice. bp b 0.05 vs. fructose-fed WT mice. cp b 0.05 vs. water-fed PPARβ/δ−/−mice.

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galactose N-acetyl-galactosamine-specific lectin 1 (Mgl1), we observedthat the expression of bothmarkerswas significantly reduced in PPARβ/δ-deficient mice compared to wild-type mice, which is consistentwith the results of Kang et al. (2008) [20] (Fig. 3F and G). However,fructose-feeding did not exacerbate these changes, making it unlikelythat polarization of macrophages was responsible for the increase ininflammation and glucose intolerance observed in the fructose-fedPPARβ/δ-deficient mice.

3.4. Increased activation of the oxLDL-CD36-JNK pathway in white adiposetissue from fructose-fed PPARβ/δ-deficient mice

Given that oxLDL promotes a CD36-dependent insulin resistancephenotype in adipocyteswhich involves inflammation andmacrophageinfiltration in adipose tissue [11], we evaluated serum oxLDL levels.Mice deficient in PPARβ/δ exhibited a significant increase in serumlevels of oxLDL compared to wild-type mice, whereas fructose intakedid not exacerbate the levels of oxLDL (Fig. 4A). In agreement withthis, the protein levels of the lectin-like ox-LDL receptor-1 (LOX-1), areceptor up-regulated by oxLDL [24], were increased similarly inwater and fructose fed PPARβ/δ deficient mice (Fig. 4B). Activation ofthe oxLDL-CD36-JNK pathway depends on CD36, since it has beenreported that the absence of this scavenger receptor protects miceagainst high-fat diet-induced inflammation and insulin resistance [11].When we measured CD36 protein levels, we observed an increase inwater-fed PPARβ/δ-deficient mice compared to wild-type mice fed

with either water or fructose (Fig. 4C). This suggests that the absenceof this nuclear receptor increases CD36 protein levels in adipose tissue.It is worth noting that when the deficiency of PPARβ/δ was combinedwith fructose feeding, a significant increase was observed compared towater-fed PPARβ/δ-deficient mice, suggesting that fructose intakecauses an additional increase in CD36 protein levels in these mice.Interestingly, only fructose-fed PPARβ/δ-deficient mice exhibited asignificant increase in Cd36 mRNA levels (Fig. 4D), suggesting that theadditional increase in CD36 protein levels observed in this groupmight involve a transcriptional mechanism. In agreement with thechanges in CD36 protein levels, JNK2 phosphorylation was increasedin water-fed PPARβ/δ-deficient mice and this increase was significantlyhigher in fructose-fed PPARβ/δ-deficientmice (Fig. 4E). Consistent withthis, the fructose-fed PPARβ/δ-deficient mice displayed an increase inphospho-c-Jun levels (Fig. 4F), a downstream target of JNK, as well asin the expression of activated transcription factor 3 (Atf-3) (Fig. 4G), agenewhich is under the control of the JNK-activated transcription factoractivator protein 1 (AP-1) [25]. Although Atf3 expression is also en-hanced following endoplasmic reticulum (ER) stress, no increase wasobserved in other markers of this process, such as glucose-regulatedprotein 78 (Grp78/Bip) or CCAAT/enhancer-binding protein homolo-gous protein (Chop) mRNA levels (data not shown). This suggests thatthe increase in Atf3 expression was not the result of an increase in ERstress. Overall, these findings suggest that, although PPARβ/δ-deficiency leads to enhanced oxLDL levels, only the combination ofthis genotype with fructose intake reaches CD36 protein levels

Fig. 2. Fructose increases adipocyte hypertrophy. A, representative microphotographs ofhematoxylin and eosin (H&E) staining of white adipose tissue. B, quantification of adipo-cyte size. Data are expressed as frequency (%). Results are presented as mean ± S.E.Adipocyte diameters were measured in five random fields of adipose tissue sectionsfrom three mice per group. The total adipocyte number was 500 per group.

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sufficient to activate JNK1/2, which in turn might increase the expres-sion of inflammatory markers.

3.5. PPARβ/δ activation prevents the fructose-induced increase in CD36protein levels and insulin signaling

Since CD36 is crucial in the oxLDL-mediated activation of JNK, in-flammation and insulin resistance in adipocytes [11,26], we next stud-ied the mechanisms responsible for the effects of fructose and PPARβ/δ on CD36 protein levels. We took advantage of the human SGBS adipo-cytes. These cells have a high capacity for adipose differentiation and,therefore, represent a unique tool for studying human fat cell develop-ment andmetabolism [17].Whenwe examined the CD36 protein levels,we observed that adipocytes exposed to 25 mM fructose exhibited anincrease in protein levels compared to those in isosmotic control exper-iments carried out with mannitol (Fig. 5A). This increase was slightlyhigher in cells exposed to fructose plus the PPARβ/δ antagonistGSK0660, although the differences did not reach statistical significance,when compared to fructose alone. However, when cells were exposedto fructose in the presence of the PPARβ/δ activator GW501516, CD36protein levels were reduced even below the control levels. AlthoughPPARγ is a master regulator of Cd36 [27], the changes induced by

fructose seemed not to be related to this nuclear receptor, since nochanges were observed in PPARγ protein levels following fructose ex-posure. Then we examined whether PPARβ/δ activation by GW501516was able to prevent fructose-induced insulin resistance by assessinginsulin-stimulated AKT phosphorylation. Exposure to fructose reducedinsulin-stimulated AKT phosphorylation compared to that of cellsexposed only to mannitol (Fig. 5B). Notably, cells exposed to fructoseco-incubated with GW501516 displayed an important recovery of AKTphosphorylation. Similar results were obtained with murine 3T3-L1adipocytes (Fig. 5C). Next, we evaluated the potential mechanism bywhich PPARβ/δ activation might increase AKT phosphorylation andprevent the increase in CD36 caused by fructose. Since previous studieshave reported that activation of the transcription factor Nrf2upregulates Cd36 expression in 3T3-L1 cells [12], we explored the con-tribution of this transcription factor to the changes in CD36 proteinlevels observed in our conditions by using theNrf2 inhibitor trigonelline[28]. Nrf2 activation seems to play an important role in fructose-induced insulin resistance in adipocytes since inhibition of this tran-scription factor with trigonelline completely prevented the decrease ininsulin-stimulated AKT phosphorylation caused by exposure to thiscarbohydrate (Fig. 5D). Interestingly, similarly to trigonelline, thePPARβ/δ agonist GW501516 abolished the increase in Nrf2 caused byfructose in adipocytes, suggesting that PPARβ/δ activation reducesCD36 protein levels in fructose-exposed adipocytes by preventing theincrease in nuclear Nrf2 levels (Fig. 5E). This protein is usually detectedas a dimer of 90–110 kDa. However, in our 3T3-L1 adipocytes Nrf2 wasdetected at 56 kDa in agreementwith a previous study [29]. In addition,Nrf2 inhibition by trigonelline abolished the increase in CD36 proteinlevels caused by fructose in adipocytes (Fig. 5F), confirming thatfructose-induced CD36 up-regulation involves Nrf2. In agreementwith the changes in Nrf2, the protein levels of the well-describedNrf-2 target gene NAD(P)H:quinone oxidoreductase 1 (Nqo1) [30]were increased by fructose and this increase was prevented by co-incubation with trigonelline (Fig. 5G). Finally, we examined the proteinlevels of Nrf2 and NQO1 in mice. Phosphorylated levels of Nrf2 are anindicator of the activity of this transcription factor [31] andwe observedthat Nrf2was activated in thewhite adipose tissue of PPARβ/δ-deficientmice since enhanced nuclear levels of phosphorylated Nrf2 (~110 kDa)were detected (Fig. 5H). Similarly, NQO1 protein levels were increasedonly in the white adipose tissue of PPARβ/δ-deficientmice and that thisincrease was exacerbated by fructose feeding (Fig. 5I). Moreover, theoxidative stress status measured by the lipid peroxidized productMDA was increased in fructose-fed mice, but no significant differenceswere observed between fructose-fed wild-type and PPARβ/δ-deficientmice (Fig. 5J).

4. Discussion

According to the steps proposed in the development of adiposetissue inflammation and insulin resistance [32], hypertrophic adipo-cytes initially begin to secrete low levels of TNF-α, which stimulatepreadipocytes to produce MCP-1 [33]. As a result of the increase inMCP-1, macrophages infiltrate into adipose tissue. Macrophages, alongwith adipocytes and other cells, drive a vicious cycle of macrophage in-filtration and pro-inflammatory cytokine production. These inflamma-tory cytokines, such as TNF-α, activate JNK and IKK serine-kinases,which promote phosphorylation of IRS-1 on Ser307 (Ser312 in humanIRS-1), leading to inhibition of insulin receptor signaling and insulin re-sistance. A new recent player in this scenario is CD36 [11,32,34]. Severalpathological ligands, including oxLDL, can signal via CD36 and therebyaffect both inflammatory and insulin signaling pathways in adipocytes.Our findings suggest that fructose intake in PPARβ/δ-deficient miceleads to adipocyte hypertrophy, which is accompanied by increased in-flammation and macrophage infiltration, and increased glucose intoler-ance through a mechanism that could involve over activation of theoxLDL-CD36-JNK pathway. Of note, our findings also point to Nrf2 as a

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Fig. 4. Increased activation of the oxLDL-CD36-JNK pathway in fructose-fed PPARβ/δ-deficient mice. A, oxLDL serum levels. Analysis of LOX-1 (B) and CD36 (C) protein levels by immu-noblotting. D, Cd36mRNA levels. Cell lysates were assayed byWestern-blot analysiswith antibodies against total and phospho-JNK1/2 (E) and total and phosphor-c-Jun (F). Immunoblotsfrom four separate experiments were quantified and are presented in the corresponding bar graphs. G, Atf-3 mRNA levels. Graphs represent the quantification of the Aprt-normalizedmRNA levels. Data are expressed as mean ± S.D. (6 mice per group). ap b 0.05 vs. water-fed WT mice. bp b 0.05 vs. fructose-fed WT mice. cp b 0.05 vs. water-fed PPARβ/δ−/−mice.

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key transcription factor involved in fructose-induced CD36 levels andinsulin resistance in adipocytes.

The findings of this study show that feeding fructose to PPARβ/δ-deficient mice led to an increase in weight gain and hepatic steatosis,whichwas not observed in wild-type animals. We do not know the rea-son for the lack of effects of fructose on these parameters in wild-typeanimals, although the different genetic background (C57BL/6X129/SV)compared to previous studies (C57BL/6 background) and age could beinvolved, since a recent study reported that fructose feeding in youngC57BL/6 mice did not negatively affect final body weight [35]. Theemergence of these effects in fructose-fed PPARβ/δ-deficient mice sug-gests that the presence of this nuclear receptor protects against thesephenotypic manifestations. The lack of an effect of fructose on hepaticsteatosis did not extend to other tissues, such as white adipose tissue,thereby confirming that the treatment with fructose supplementationfunctioned as intended. Thus, fructose feeding elicited adipocyte

Fig. 3. Increased inflammation and macrophage infiltration in fructose-fed PPARβ/δ-deficienquantification of the Aprt-normalized mRNA levels. E, representative images of CD11b immuare expressed as mean ± S.D. (6 mice per group). ap b 0.05 vs.water-fed WT mice. bp b 0.05 v

hypertrophy in wild-type mice, which is consistent with previous stud-ies [36], and, to a greater extent, in PPARβ/δ-deficient mice. The pres-ence of larger adipocytes in the white adipose tissue of fructose-fedPPARβ/δ-deficient mice was accompanied by an increase in the expres-sion of Tnf-α andMcp-1, butwasnot in fructose-fedwild-typemice. Thissuggests that besides adipocyte hypertrophy, additional mechanismsare responsible for the increase in the expression of the genes men-tioned. Consistent with the increase in Mcp-1 expression, we observedenhanced macrophage infiltration in the white adipose tissue offructose-fed PPARβ/δ-deficient mice. This infiltration of macrophagescan also be responsible for part of the increase in the expression ofpro-inflammatory cytokines. It is worth emphasizing that MCP-1 con-tributes to macrophage infiltration into adipose tissue, insulin resis-tance and hepatic steatosis [37,38]; which are all features observed infructose-fed PPARβ/δ-deficient mice. Despite PPARβ/δ being able toregulate macrophage polarization [20], our findings rule out the

t mice. Tnf-α (A), Mcp-1 (B), F4/80 (C) and Cd68 (D) mRNA levels. Graphs represent thenohistochemistry of the white adipose tissue. Il-10 (F) and Mgl-1 (G) mRNA levels. Datas. fructose-fed WT mice. cp b 0.05 vs. water-fed PPARβ/δ−/−mice.

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polarization of macrophages as the mechanism responsible for the in-crease in inflammation and glucose intolerance in fructose-fed PPARβ/δ-deficient mice.

OxLDL activates JNK in adipocytes and disrupts insulin signaling in aCD36-dependentmanner [11]. PPARβ/δ-deficient mice fed either wateror fructose exhibited increased oxLDL levels, suggesting that PPARβ/δdeficiency increases these modified lipoproteins. As far as we know,this is the first study to report that PPARβ/δ deficiency increasesoxLDL and reveals a new pharmacological target that could be used toreduce levels of oxLDL, which are strongly associatedwith atherosclero-sis, and diabetes and its complications [39]. Despite it having beenreported that fructose feeding can increase the plasma levels of oxLDLin humans [2,40], we did not observe an additional increase infructose-fed vs. water-fed PPARβ/δ-deficient mice.

CD36 is considered a PPARβ/δ target gene [41], although activationof PPARβ/δ may yield different tissue-specific results. Thus, it has beenreported that activation of PPARβ/δ results in enhanced CD36 levels inskeletal muscle [42]; whereas in liver, PPARβ/δ agonists reduce Cd36expression [41]. In agreement with this PPARβ/δ-dependent regulationof CD36, water-fed PPARβ/δ-deficient mice showed enhanced CD36protein levels in white adipose tissue. Interestingly, fructose-fedPPARβ/δ-deficient animals exhibited significantly higher CD36 proteinlevels than water-fed mice. This fructose-induced increase is consistentwith a previous study that reported that fructose intake (60% for8 weeks) increases CD36 protein levels in the white adipose tissue ofrats [43]. Our in vitro studies in SGBS adipocytes confirmed that fructoseincreases CD36 protein levels.

The additional increase in CD36 protein levels resulting fromfructose in PPARβ/δ-null mice compared to water-fed PPARβ/δ-nullmice seems to be dependent on the transcriptional activation of theCd36 gene, since Cd36 mRNA levels were increased in the former.Since it has been reported that Nrf2 increases Cd36 expression in 3T3-L1 cells [12], we focused on this transcription factor. Our in vitrofindingsconfirmed that fructose increased the nuclear protein levels of Nrf2and that the increase in CD36 protein levels caused by fructose was de-pendent on this transcription factor, inasmuch as the Nrf2 inhibitortrigonelline reversed the increase. Notably, the PPARβ/δ agonistGW501516 completely abolished the increase in Nrf2 induced byfructose, which suggests that the lack of PPARβ/δ may contribute to anincrease in Nrf2 transcriptional activity. In agreement with this, whiteadipose tissue from PPARβ/δ-null mice showed enhanced phosphory-lated protein levels of Nrf2 and of its target gene Nqo1 and the increaseof the latter was higher in those mice fed fructose than in those fedwater, suggesting that the lack of PPARβ/δ and fructose synergisticallycontribute to increase CD36 levels through Nrf2. Notwithstanding theforegoing, we did not observe a higher increase in phosphorylatedNrf2 levels in fructose-fed PPARβ/δ-deficient compared to water-fedPPARβ/δ-deficient mice. A similar behavior has been described previ-ously in 3T3-L1 cells, where oxLDL treatment increased CD36 proteinlevels for 5 days, whereas nuclear Nrf2 levels peaked at 5 h followingoxLDL exposure, but returned to basal levels at 7 h [12]. We do notknow the reason for this, but it has been reported that CD36 inhibitsNrf2 nuclear localization [44]. Thus, sustained increase of CD36 in vivomight activate this mechanism, causing a reduction in nuclear Nrf2proteins.

Our findings also support an important role for Nrf2 in fructose-induced insulin resistance in adipocytes, since it was completelyreversed by the Nrf2 inhibitor trigonelline. In fact, Nrf2-null mice havedecreased white adipose tissue mass and smaller adipocyte formation

Fig. 5. PPARβ/δ-activation prevents the increase in CD36 protein levels in adipocytes caused25 mM fructose or mannitol with or without 10 μmol/l GW501516, 10 μmol/l GSK0660 or 0.5for the last 10min. Cell lysates from human SGBS adipocytes were assayed forWestern-blot anaAKT (Ser473) (B) in human SGBS. Cell lysates frommurine 3T3-L1 adipocytes were assayed fornuclear Nrf2 (E), CD36 (F) and NQO1 (G). Analysis of Nrf2 (H) and NQO1 protein levels by immmice (PPARβ/δ−/−) fed with either water or water containing 30% fructose. ap b 0.05 vs.water-

and protection against weight gain and obesity than wild-type mice[13]. Consistent with this, it has been reported that enhanced Nrf2activity exacerbates insulin resistance in leptin-deficient mice [45].

Given that CD36 is key for the activation of JNK in adipocytes byoxLDL and for the disruption of insulin signaling [11], the differencesin CD36 protein levels in water-fed and fructose-fed PPARβ/δ-nullmice may result in enhanced metabolic alterations. Thus, althoughthe levels of oxLDL were similar in both water-fed and fructose-fedPPARβ/δ-deficient mice, the higher increase in CD36 protein levels inthe transgenic mice receiving fructose, might entail a higher amplifica-tion of the oxLDL-CD36-JNK pathway. Consistentwith this, we observedincreased JNK2phosphorylation in thewhite adipose tissue of PPARβ/δ-deficient mice fed with fructose, which resulted in an increase of itsdownstream targets (phospho-c-jun and Atf3 mRNA levels). Althoughthe JNK1 isoform has primarily been implicated in the development ofobesity and insulin resistance [46], JNK2 might also play a role as thebalance between JNK1 and JNK2, which influences total JNK activity,and is a critical determinant of inflammatory cytokine production [47].

Although we have used a human adipocytic cell line to know therelevance of the data obtained in murine models, one limitation of thisstudy is the lack of data from human samples to assess whether themechanism described in this study also operates in human beings andcontributes to exacerbating metabolic alterations.

In summary, our findings show that glucose intolerance in PPARβ/δ-deficient mice is associated with the generation of oxLDL and increasedCD36 protein levels in white adipose tissue. When this genotype wascombined with a chronic intake of fructose, increases in adipocytehypertrophy, inflammation, macrophage infiltration and glucoseintolerance were observed. Since previous studies have reported thatPPARβ/δ activity might be reduced in the white adipose tissue ofobese insulin-resistant patients [10], it might be interesting to studywhether inflammation and development of type 2 diabetes mellitus isaccelerated in obese high fructose consumers through the mechanismreported in this study.

Supplementary data to this article can be found online at http://10.1016/j.bbadis.2015.02.010.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgments

We are grateful to Liliane Michalik (University of Lausanne) forreading and discussing themanuscript.Wewould like to thank theUni-versity of Barcelona's Language Advisory Service for its help. This workwas funded by the Spanish Ministerio de Economía y Competitividad(SAF2009-06939 and SAF2012-30708), the Fondo de InvestigaciónSanitaria (FIS) (PI08/0733, PI11/00049 and PS09/00997) and theEuropean Union ERDF funds. L.S. is supported by an FPI grant from theSpanishMinisterio de Economía y Competitividad. M.R.CH. is supportedby the Research Stabilization Programme of the Instituto de Salud CarlosIII (ISCIII) co-financed by Institut Català de Salut (ICS) in Catalonia.CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)is an initiative of the ISCIII (Ministerio de Economía y Competitividad ofSpain).

by fructose exposure. Human SGBS or murine 3T3-L1 adipocytes were exposed to eitherμmol/l trigonelline for 24 h. When indicated cells were incubated with 100 nmol/l insulinlysis with antibodies against CD36, PPARγ and β-actin (A) and against total and phospho-Western-blot analysis with antibodies against total and phospho-AKT (Ser473) (C and D),unoblotting (I) andMDA (J) from adipose tissue of wild-type (WT) and PPARβ/δ-deficientfedWTmice. bp b 0.05 vs. fructose-fedWTmice. cp b 0.05 vs.water-fed PPARβ/δ−/−mice.

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References

[1] M. Collino, High dietary fructose intake: sweet or bitter life? World J. Diabetes 2(2011) 77–81.

[2] K.L. Stanhope, J.M. Schwarz, N.L. Keim, S.C. Griffen, A.A. Bremer, J.L. Graham, et al.,Consuming fructose-sweetened, not glucose-sweetened, beverages increasesvisceral adiposity and lipids and decreases insulin sensitivity in overweight/obesehumans, J. Clin. Invest. 119 (2009) 1322–1334.

[3] L. Tappy, K.A. Lê, Metabolic effects of fructose and theworldwide increase in obesity,Physiol. Rev. 90 (2010) 23–46.

[4] J. Hallfrisch, Metabolic effects of dietary fructose, FASEB J. 4 (1990) 2652–2660.[5] N.K. Pollock, V. Bundy, W. Kanto, C.L. Davis, P.J. Bernard, H. Zhu, et al., Greater

fructose consumption is associated with cardiometabolic risk markers and visceraladiposity in adolescents, J. Nutr. 142 (2012) 251–257.

[6] G.D. Barish, V.A. Narkar, R.M. Evans, PPAR delta: a dagger in the heart of themetabolic syndrome, J. Clin. Invest. 116 (2006) 590–597.

[7] E. Bedu, W. Wahli, B. Desvergne, Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases, Expert Opin. Ther. Targets 9(2005) 861–873.

[8] L. Salvadó, L. Serrano-Marco, E. Barroso, X. Palomer, M. Vázquez-Carrera, TargetingPPARβ/δ for the treatment of type 2 diabetes mellitus, Expert Opin. Ther. Targets16 (2012) 209–223.

[9] L. Andrulionyte, P. Peltola, J.L. Chiasson, M. Laakso, STOP-NIDDM Study Group.Single nucleotide polymorphisms of PPARD in combination with the Gly482Ser sub-stitution of PGC-1A and the Pro12Ala substitution of PPARG2 predict the conversionfrom impaired glucose tolerance to type 2 diabetes: the STOP-NIDDM trial, Diabetes55 (2006) 2148–2152.

[10] L. Serrano-Marco, M.R. Chacón, E. Maymó-Masip, E. Barroso, L. Salvadó, M.Wabitsch, et al., TNF-α inhibits PPARβ/δ activity and SIRT1 expression throughNF-κB in human adipocytes, Biochim. Biophys. Acta 1821 (2012) 1177–1185.

[11] D.J. Kennedy, S. Kuchibhotla, K.M. Westfall, R.L. Silverstein, R.E. Morton, M.A.Febbraio, CD36-dependent pathway enhances macrophage and adipose tissueinflammation and impairs insulin signaling, Cardiovasc. Res. 89 (2011) 604–613.

[12] M. D'Archivio, B. Scazzocchio, C. Filesi, R. Varì, M.T. Maggiorella, L. Sernicola, et al.,Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1preadipocytes, FEBS Lett. 582 (2008) 2291–2298.

[13] J. Pi, L. Leung, P. Xue, W. Wang, Y. Hou, D. Liu, et al., Deficiency in the nuclear factorE2-related factor-2 transcription factor results in impaired adipogenesis andprotects against diet-induced obesity, J. Biol. Chem. 285 (2010) 9292–9300.

[14] K. Nadra, S.I. Anghel, E. Joye, N.S. Tan, S. Basu-Modak, D. Trono, et al., Differentiationof trophoblast giant cells and their metabolic functions are dependent onperoxisome proliferator-activated receptor beta/delta, Mol. Cell. Biol. 26 (2006)3266–3281.

[15] A. Spruss, G. Kanuri, K. Uebel, S.C. Bischoff, I. Bergheim, Role of the inducible nitricoxide synthase in the onset of fructose-induced steatosis in mice, Antioxid. RedoxSignal. 14 (2011) 2121–2135.

[16] A. Spruss, G. Kanuri, S. Wagnerberger, S. Haub, S.C. Bischoff, I. Bergheim, Toll-likereceptor 4 is involved in the development of fructose-induced hepatic steatosis inmice, Hepatology 50 (2009) 1094–1104.

[17] M.Wabitsch, R.E. Brenner, I. Melzner, M. Braun, P. Moller, E. Heinze, et al., Character-ization of a human preadipocyte cell strain with high capacity for adipose differen-tiation, Int. J. Obes. Relat. Metab. Disord. 25 (2001) 8–15.

[18] L. Salvadó, T. Coll, A.M. Gómez-Foix, E. Salmerón, E. Barroso, X. Palomer, et al., Oleateprevents saturated-fatty-acid-induced ER stress, inflammation and insulinresistance in skeletal muscle cells through an AMPK-dependent mechanism,Diabetologia 56 (2013) 1372–1382.

[19] M. Lönn, K. Mehlig, C. Bengtsson, L. Lissner, Adipocyte size predicts incidence of type2 diabetes in women, FASEB J. 24 (2010) 326–331.

[20] K. Kang, S.M. Reilly, V. Karabacak, M.R. Gangl, K. Fitzgerald, B. Hatano, et al.,Adipocyte-derived Th2 cytokines and myeloid PPARdelta regulate macrophagepolarization and insulin sensitivity, Cell Metab. 7 (2008) 485–495.

[21] M. Jernås, J. Palming, K. Sjöholm, E. Jennische, P.A. Svensson, B.G. Gabrielsson, et al.,Separation of human adipocytes by size: hypertrophic fat cells display distinct geneexpression, FASEB J. 20 (2010) 1540–1542.

[22] T. Skurk, C. Alberti-Huber, C. Herder, H. Hauner, Relationship between adipocytesize and adipokine expression and secretion, J. Clin. Endocrinol. Metab. 92 (2007)1023–1033.

[23] C.N. Lumeng, J.L. Bodzin, A.R. Saltiel, Obesity induces a phenotypic switch in adiposetissue macrophage polarization, J. Clin. Invest. 117 (2007) 175–184.

[24] T. Sawamura, N. Kume, T. Aoyama, H. Moriwaki, H. Hoshikawa, Y. Aiba, et al., Anendothelial receptor for oxidized low-density lipoprotein, Nature 386 (1997)73–77.

[25] Y. Cai, C. Zhang, T. Nawa, T. Aso, M. Tanaka, S. Oshiro, et al., Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activationof c-Jun NH(2)-terminal kinase and promoter response element, Blood 96 (2000)2140–2148.

[26] R.L. Silverstein, M. Febbraio, CD36, a scavenger receptor involved in immunity,metabolism, angiogenesis, and behavior, Sci. Signal. 2 (2009) re3.

[27] C. Capurso, A. Capurso, From excess adiposity to insulin resistance: the role of freefatty acids, Vasc. Pharmacol. 57 (2012) 91–97.

[28] A. Arlt, S. Sebens, S. Krebs, C. Geismann, M. Grossmann, M.L. Kruse, et al., Inhibitionof the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancercells more susceptible to apoptosis through decreased proteasomal gene expressionand proteasome activity, Oncogene 32 (2013) 4825–4835.

[29] E.E. Vomhof-Dekrey, M.J. Picklo, NAD(P)H:quinone oxidoreductase 1 activityreduces hypertrophy in 3T3-L1 adipocytes, Free Radic. Biol. Med. 53 (2012)690–700.

[30] A.T. Dinkova-Kostova, P. Talalay, NAD(P)H:quinone acceptor oxidoreductase 1(NQO1), a multifunctional antioxidant enzyme and exceptionally versatilecytoprotector, Arch. Biochem. Biophys. 501 (2010) 116–123.

[31] I. Lázaro, R. Ferré, L. Masana, A. Cabré, Akt and ERK/Nrf2 activation by PUFAoxidation-derived aldehydes upregulates FABP4 expression in human macro-phages, Atherosclerosis 230 (2013) 216–222.

[32] H. Xu, G.T. Barnes, Q. Yang, G. Tan, D. Yang, C.J. Chou, et al., Chronic inflammation infat plays a crucial role in the development of obesity-related insulin resistance, J.Clin. Invest. 112 (2003) 1821–1830.

[33] H.T. Nicholls, G. Kowalski, D.J. Kennedy, S. Risis, L.A. Zaffino, N. Watson, et al.,Hematopoietic cell-restricted deletion of CD36 reduces high-fat diet-inducedmacrophage infiltration and improves insulin signaling in adipose tissue, Diabetes60 (2011) 1100–1110.

[34] L. Cai, Z. Wang, A. Ji, J.M. Meyer, D.R. van der Westhuyzen, Scavenger receptor CD36expression contributes to adipose tissue inflammation and cell death in diet-induced obesity, PLoS One 7 (2012) e36785.

[35] E.K. Tillman, D.A. Morgan, K. Rahmouni, S.J. Swoap, Three months of high-fructosefeeding fails to induce excessive weight gain or leptin resistance in mice, PLoSOne 9 (2014) e107206.

[36] J.P. Fariña, M.E. García, A. Alzamendi, A. Giovambattista, C.A. Marra, E. Spinedi, et al.,Antioxidant treatment prevents the development of fructose-induced abdominaladipose tissue dysfunction, Clin. Sci. 125 (2013) 87–97.

[37] H. Kanda, S. Tateya, Y. Tamori, K. Kotani, K. Hiasa, R. Kitazawa, et al., MCP-1 contrib-utes to macrophage infiltration into adipose tissue, insulin resistance, and hepaticsteatosis in obesity, J. Clin. Invest. 116 (2006) 1494–1505.

[38] N. Kamei, K. Tobe, R. Suzuki, M. Ohsugi, T. Watanabe, N. Kubota, et al., Overexpres-sion of monocyte chemoattractant protein-1 in adipose tissues causes macrophagerecruitment and insulin resistance, J. Biol. Chem. 281 (2006) 26602–26614.

[39] F. Piarulli, A. Lapolla, G. Sartore, C. Rossetti, G. Bax, M. Noale, et al., Autoantibodiesagainst oxidized LDLs and atherosclerosis in type 2 diabetes, Diabetes Care 28(2005) 653–657.

[40] K.L. Stanhope, P.J. Havel, Fructose consumption: recent results and their potentialimplications, Ann. N. Y. Acad. Sci. 1190 (2010) 15–24.

[41] J.M. Ye, J. Tid-Ang, N. Turner, X.Y. Zeng, H.Y. Li, G.J. Cooney, et al., PPARδ agonistshave opposing effects on insulin resistance in high fat-fed rats and mice due todifferent metabolic responses in muscle, Br. J. Pharmacol. 163 (2011) 556–566.

[42] J. Cresser, A. Bonen, A. Chabowski, L.E. Stefanyk, R. Gulli, I. Ritchie, et al., Oraladministration of a PPAR-delta agonist to rodents worsens, not improves, maximalinsulin-stimulated glucose transport in skeletal muscle of different fibers, Am. J.Physiol. Regul. Integr. Comp. Physiol. 299 (2010) R470–R479.

[43] B. Qin, M.M. Polansky, R.A. Anderson, Cinnamon extract regulates plasma levels ofadipose-derived factors and expression of multiple genes related to carbohydratemetabolism and lipogenesis in adipose tissue of fructose-fed rats, Horm. Metab.Res. 42 (2010) 187–193.

[44] W. Li, M. Febbraio, S.P. Reddy, D.Y. Yu, M. Yamamoto, R.L. Silverstein, CD36participates in a signaling pathway that regulates ROS formation in murineVSMCs, J. Clin. Invest. 120 (2010) 3996–4006.

[45] J. Xu, S.R. Kulkarni, A.C. Donepudi, V.R. More, A.L. Slitt, Enhanced Nrf2 activityworsens insulin resistance, impairs lipid accumulation in adipose tissue,and increases hepatic steatosis in leptin-deficient mice, Diabetes 61 (2012)3208–3218.

[46] J. Hirosumi, G. Tuncman, L. Chang, C.Z. Görgün, K.T. Uysal, K. Maeda, et al., A centralrole for JNK in obesity and insulin resistance, Nature 420 (2002) 333–336.

[47] G. Tuncman, J. Hirosumi, G. Solinas, L. Chang, M. Karin, G.S. Hotamisligil, Functionalin vivo interactions between JNK1 and JNK2 isoforms in obesity and insulinresistance, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 10741–10746.