Orla M. Finucane,1 Claire L. Lyons,1 Aoife M. Murphy,1 Clare M. Reynolds,1

Rut Klinger,2 Niamh P. Healy,1 Aoife A. Cooke,1 Rebecca C. Coll,3 Liam McAllan,4

Kanishka N. Nilaweera,4 Marcella E. O’Reilly,1 Audrey C. Tierney,5 Melissa J. Morine,6

Juan F. Alcala-Diaz,7 Jose Lopez-Miranda,7 Darran P. O’Connor,2 Luke A. O’Neill,3

Fiona C. McGillicuddy,1 and Helen M. Roche1

Monounsaturated Fatty Acid–EnrichedHigh-Fat Diets Impede Adipose NLRP3Inflammasome–Mediated IL-1bSecretion and Insulin ResistanceDespite ObesityDiabetes 2015;64:2116–2128 | DOI: 10.2337/db14-1098

Saturated fatty acid (SFA) high-fat diets (HFDs) enhanceinterleukin (IL)-1b–mediated adipose inflammation andinsulin resistance. However, the mechanisms by whichdifferent fatty acids regulate IL-1b and the subsequenteffects on adipose tissue biology and insulin sensitivityin vivo remain elusive. We hypothesized that the re-placement of SFA for monounsaturated fatty acid(MUFA) in HFDs would reduce pro-IL-1b priming in ad-ipose tissue and attenuate insulin resistance via MUFA-driven AMPK activation. MUFA-HFD–fed mice displayedimproved insulin sensitivity coincident with reduced pro-IL-1b priming, attenuated adipose IL-1b secretion, andsustained adipose AMPK activation compared withSFA-HFD–fed mice. Furthermore, MUFA-HFD–fed micedisplayed hyperplastic adipose tissue, with enhancedadipogenic potential of the stromal vascular fractionand improved insulin sensitivity. In vitro, we demon-strated that the MUFA oleic acid can impede ATP-induced IL-1b secretion from lipopolysaccharide- andSFA-primed cells in an AMPK-dependent manner.Conversely, in a regression study, switching fromSFA- to MUFA-HFD failed to reverse insulin resistancebut improved fasting plasma insulin levels. In humans,

high-SFA consumers, but not high-MUFA consumers,displayed reduced insulin sensitivity with elevatedpycard-1 and caspase-1 expression in adipose tissue.These novel findings suggest that dietary MUFA canattenuate IL-1b–mediated insulin resistance and adi-pose dysfunction despite obesity via the preservationof AMPK activity.

Intricate cellular mechanisms contribute to obesity andtype 2 diabetes (T2D), wherein the development ofinsulin resistance (IR) is critical (1). Obesity-associatedIR reflects sophisticated networks interlinking metabolicand inflammatory processes; a phenomenon aptly coined“meta-inflammation” where the nucleotide binding andoligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a key regulatory hub.However, the mechanisms by which different fatty acidsregulate adipose tissue NLRP3 inflammasome activity andmetabolic dysfunction remain ill defined.

Recent studies (2–5) have established a critical role forthe NLRP3 inflammasome and interleukin (IL)-1b in

1Nutrigenomics Research Group, Conway Institute of Biomedical and Biomolec-ular Research, and Institute of Food and Health, University College Dublin, Bel-field, Dublin, Ireland2School of Biomolecular & Biomedical Science, University College Dublin, Belfield,Dublin, Ireland3Inflammatory Research Group, Biomedical Sciences Institute, Trinity CollegeDublin, Dublin, Ireland4Teagasc Food Research Centre, Moorepark, Fermoy, Ireland5Department of Dietetics and Human Nutrition, La Trobe University, Melbourne,Victoria, Australia6The Microsoft Research–University of Trento Centre for Computational and Sys-tems Biology, Rovereto, Italy

7Lipids and Atherosclerosis Research Unit, Reina Sofía University Hospital,and CIBER Phyisiopathology of Obesity and Nutrition (CIBEROBN), University ofCórdoba, Córdoba, Spain

Corresponding author: Helen M. Roche, helen.roche@ucd.ie.

Received 22 July 2014 and accepted 14 January 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1098/-/DC1.

© 2015 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.

2116 Diabetes Volume 64, June 2015

OBESITY

STUDIES

peripheral IR and T2D. Furthermore, IL-1b impedes adi-pocyte insulin signaling (6) and adipogenesis (7). Giventhe highly potent nature of IL-1b, release is tightly regu-lated and requires two signals. First, the cell is “primed” toproduce pro-IL-1b via Toll-like receptor 4 (TLR4)/nuclearfactor-kB (8). The NLRP3 inflammasome, a lipid-responsiveprotein complex, then mediates the caspase-1–dependentprocessing of pro-IL-1b to mature IL-1b (9). It hasbeen established that saturated fatty acids (SFAs), partic-ularly palmitic acid (PA), can initiate the first “priming”signal via TLR4 (5). The second signal is likely mediatedby danger-associated molecular pattern molecules such asATP, uric acid, and reactive oxygen species (10). The roleof monounsaturated fatty acids (MUFAs) in inflammationand IR remains contentious. Human studies (11,12) re-port conflicting evidence regarding the cardioprotectiverole of MUFA-rich diets. Mechanistic evidence regardingthe effects of different fatty acids on IL-1–mediated in-flammation and insulin sensitivity in adipose tissue biol-ogy in vivo is lacking.

This present study shows that a MUFA high-fat diet(HFD) does not prime pro-IL-1b in adipose tissue con-comitant with enhanced AMPK activation, a hyperplas-tic adipose morphology, and partial protection from IR.Furthermore, MUFAs can impede ATP-induced IL-1bsecretion from lipopolysaccharide (LPS)-primed cellsin an AMPK-dependent manner in vitro. Additionally,high dietary SFA intake, but not high MUFA intake, isassociated with IR and inflammation in humans with T2D.This study demonstrates that dietary MUFAs can attenuateIL-1b–mediated IR and adipose tissue dysfunction despiteobesity.

RESEARCH DESIGN AND METHODS

MaterialsCell culture material was purchased from Lonza (Slough,U.K.). All other reagents, unless otherwise stated, werefrom Sigma-Aldrich Ireland Ltd. (Wicklow, Ireland).

AnimalsC57BL/6 male mice (8–9 weeks of age) were purchasedfrom Harlan U.K. Ltd. Ethical approval was obtained fromthe University College Dublin Ethics Committee, and micewere maintained according to European Union and IrishDepartment of Health regulations. Two feeding studieswere completed. 1) Mice were fed SFA-HFD (45% kcalPA), MUFA-HFD (45% kcal oleic acid [OA]) (ResearchDiets Inc., New Brunswick, NJ), or standard chow diet(5.2% fat: 0.9% SFA, 1.3% MUFA, 3.4% PUFA) (Teklad;Harlan U.K. Ltd.) ad libitum for 24 weeks (dietary com-position is outlined in Supplementary Table 1). Fasted (6 h)mice were injected with either saline or insulin (1.5 units/kg;Actrapid; Novo Nordisk, Bagsvaerd, Denmark) 15 min priorto euthanasia by cervical dislocation. 2) Mice were fedSFA-HFD for 16 weeks and were either maintained onan SFA-HFD or switched to a MUFA-HFD for a further16 weeks.

Intraperitoneal Glucose and Insulin Tolerance TestFasted mice (6 h) were injected intraperitoneally with25% (weight for volume) glucose (1.5 g/kg) (B. BraunMedical Ltd., Dublin, Ireland) or insulin (0.5 units/kg).Blood glucose levels were measured before and aftera glucose/insulin challenge. In a separate procedure,overnight-fasted mice were administered glucose (1.5 g/kg),and blood samples were collected by tail vein bleedsampling. Plasma insulin levels were determined usingan ultra-sensitivity insulin ELISA (Crystal Chem Inc.,Downers Grove, IL).

Stromal Vascular Fraction CultureTo separate the stromal vascular fraction (SVF) from theadipocyte fraction, epididymal adipose tissue was mincedand collagenase (2 mg/mL) digested prior to centrifuga-tion. The adipocyte (200 mL packed volume/mL) and SVFwere seeded (1 3 106 cells/mL), cultured in completemedia (DMEM, 10% FBS, and 1% penicillin/streptomycin),and treated with or without ATP (5 mmol/L) for 24 h, orwith or without LPS (10 ng/mL) for 3 h. Protein lysatesand culture media were harvested for further analysis.Separately, the SVF was cultured for 7 days to removenonadherent cells, promoting a preadipocyte-enrichedSVF, and then was incubated with adipogenic media(10% FBS, 0.5 mmol/L isobutylmethylxanthine, 1 mmol/Ldexamethasone, and 1 mg/mL insulin) for 24 h. Cellswere harvested in TRI Reagent for gene expressionanalysis.

Bone Marrow–Derived MacrophagesBone marrow–derived cells were isolated from tibias andfibulas of mice and were cultured in media supplementedwith 30% L929 conditioned medium for 7 days prior tocell culture experiments. Bone marrow–derived macro-phages (BMMs) (1 3 106 cells/mL) were primed withLPS (10 ng/mL) for 3 h, then were treated with eitherPA or OA (250 mmol/L) for 24 h, followed by ATP(5 mmol/L) stimulation for 1 h. For NLRP3 inhibitorexperiments, preprimed BMMs were treated with the cy-tokine release inhibitory drug CRID3 (50 nmol/L) for 30min prior to ATP (5 mmol/L) stimulation for 1 h. ForAMPK inhibitor experiments, preprimed BMMs weretreated with compound C (10 mmol/L) for 1 h, incubatedwith OA (250 mmol/L) for 24 h, then stimulated withATP (5 mmol/L) for 1 h. For AMPK agonist experiments,preprimed BMMs were treated with AICAR (100 mmol/L)for 30 min, incubated with either PA or OA (250 mmol/L)for 24 h, then stimulated with ATP (5 mmol/L) for 1 h.Protein lysates and culture media were harvested for fur-ther analysis.

Lentiviral Short Hairpin RNA AMPKa1 KnockdownAMPKa1(1-4) mission lentiviral short hairpin RNA (shRNA)vector constructs (PLCO.1 [mission shRNA]) and scrambledcontrol lentiviral vectors were generated by cotransfectingwith pMD2.G and psPAX2 viral envelope and packagingvectors (courtesy of the Trono Laboratory, Lausanne,

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Switzerland) into HEK293 cells. Lentivirus-containingmedia were harvested, filtered, and used to infect humanTHP-1 monocytes. Successfully transduced cells wereselected with puromycin (0.5 mg/mL). Gene knockdownof cells was confirmed by immunoblotting.

Human StudyA subcohort of T2D subjects (n = 160–184) from theCORonary Diet Intervention With Olive Oil and Cardio-vascular PREVention (CORDIOPREV) study (NCT00924937)(13) were categorized based on tertiles of baseline fastingplasma SFA and MUFA concentrations. The associationof habitual dietary fat composition with insulin sensitivitywas determined by the insulin sensitivity index (ISI) andHOMA-IR. Baseline fasting plasma C-reactive proteinlevels were measured. This work was approved by theHospital Universitario Reina Sofía (Córdoba, Spain). In-formed consent was obtained from all subjects. The effectof dietary MUFA intervention and habitual dietary MUFAintake on insulin sensitivity and adipose gene expression ofcaspase-1 was also evaluated in the LIPGENE study and isprovided in the Supplementary Data.

General Laboratory MethodsDetailed descriptions of general methodologies, includingplasma analysis, flow cytometry, immunoblotting, andreal-time PCR, are included in the Supplementary Data.

Statistical AnalysisData are reported as the mean 6 SEM. For glucosetolerance test (GTT)/insulin tolerance test (ITT) studieswith multiple time points, we performed two-wayrepeated-measures ANOVA to test for differences inmeans between groups. When significant, post hocBonferroni-corrected t tests were applied. For the com-parison of data among three groups at a single timepoint, a one-way ANOVA was performed with post hocBonferroni-corrected t testing applied. Prism version 5(GraphPad Software Inc.) or PASW statistics version20.0 (SPSS) was used for statistical analyses. Statisticalsignificance is presented as *P , 0.05, **P , 0.01, and***P , 0.001 with respect to (w.r.t.) chow; and #P ,0.05, ##P , 0.01, and ###P , 0.001 for SFA versusMUFA in all figures.

RESULTS

HFDs EnrichedWith PA but Not OA Can Prime Pro-IL-1bProduction in the SVF of Adipose Tissue In VivoEx vivo adipose profiling showed that the SFA-HFDincreased IL-1b secretion (Fig. 1A) and active IL-1b pro-tein expression (Fig. 1B) to a greater extent than theMUFA-HFD, while tumor necrosis factor-a and IL-6 secre-tion were equivalent (data not shown). Adipose tissueapoptosis-associated speck-like protein containing CARD(ASC) protein expression was upregulated by both HFDswhile procaspase-1 protein levels were unchanged (Fig.1B). Adipose tissue NLRP3, IL-1b, and caspase-1 mRNAexpression was significantly upregulated after an SFA-HFDcompared with a MUFA-HFD, whereas the expressions of

sirtuin-1 (SIRT-1), TLR4, and IL-18 mRNA were compara-ble between the HFD groups (Fig. 1C).

The separation of adipose tissue into cellular com-partments demonstrated that the SVF from SFA-HFDmice secreted significantly higher amounts of ATP-induced IL-1b compared with MUFA-derived SVF, whileadipocytes secreted negligible levels of IL-1b (Fig. 1D).Caspase-1 is activated in response to ATP in SFA-HFD–derived, but not MUFA-HFD–derived, SVF (Fig. 1E).IL-1b, caspase-1, SIRT-1, and IL-1R1 (Supplementary Fig.1A–D) mRNA were also increased in SFA-HFD SVF com-pared with MUFA-HFD SVF. Protein levels of pro-IL-1band active IL-1b were higher after LPS stimulation inSFA-HFD SVF compared with chow and MUFA-HFDSVF (Fig. 1F). Proinflammatory M1 cell recruitment intoadipose tissue was reduced after a MUFA-HFD com-pared with an SFA-HFD (Supplementary Fig. 1E andF). In liver tissue, mRNA expression of inflammasomemarkers, and levels of phosphorylated (p) Jun NH2-terminal kinase and p-extracellular signal–related kinasewere equivalent across groups (Supplementary Fig. 2A–C).Further, there was no difference in IL-1b gene expression inskeletal muscle (Supplementary Fig. 2F).

MUFA-HFD Partially Protects Against Obesity-InducedIR and HyperinsulinemiaMUFA-HFD mice were partly protected from IR comparedwith SFA-HFD mice (Fig. 2A), despite equivalent glucosetolerance (Fig. 2B). MUFA-HFD mice had significantlylower fasting glucose and insulin concentrations, and at-tenuated insulin secretion in response to glucose chal-lenge (Supplementary Fig. 5B and Fig. 2C). Adipocyteinsulin receptor substrate (IRS)-1 and GLUT-4 mRNA ex-pression were significantly reduced by an SFA-HFD, but nota MUFA-HFD (Fig. 2D). Adipose tissue from MUFA-HFD–fedmice displayed elevated tyrosine pIRS-1 (Fig. 2E) and pAKT(Fig. 2F) levels, compared with SFA-HFD in response to in-sulin. Liver GLUT-2, IRS-1, IRS-2, G-6-P, and Pepck2 mRNAexpression and protein pAKT levels were unchanged acrossgroups (Supplementary Figs. 2D and E and 5A). Similarly inskeletal muscle, GLUT-4, IRS-1, and IRS-2 mRNA expres-sion was equivalent between HFD groups (SupplementaryFig. 2F).

MUFA-HFD mice became significantly obese comparedwith chow-fed controls, but gained less weight thanSFA-HFD mice (Supplementary Fig. 3A). Adipose tissuedepot weights were equivalent (Fig. 3C), but liver weightwas reduced in MUFA-HFD mice (Supplementary Fig.3B). MUFA-HFD mice displayed elevated energy expen-diture, with increased VO2 and heat production (Sup-plementary Fig. 3C and D), but no difference inlocomotor activity or respiratory exchange rate (data notshown). mRNA expression of markers of mitochondrialbiogenesis, including uncoupling protein-1, uncouplingprotein-2, peroxisome proliferator–activated receptor-g(PPARg) coactivator 1-a (PGC-1a), and acetyl-CoA carboxylasewere comparable between HFD groups in white and brown

2118 MUFA-HFD Impedes Adipose IL-1b Secretion and IR Diabetes Volume 64, June 2015

adipose tissue, skeletal muscle, and liver (Supplementary Fig.4A–D). No significant difference in hepatic citrate synthaseactivity was observed (Supplementary Fig. 4E).

MUFA-HFD mice maintained significantly lower ITTresults and insulin secretion when weight matched toSFA-HFD mice (Supplementary Fig. 3E and F), indicatingthat improved insulin sensitivity was independent ofbody weight. Fasting plasma leptin and adiponectin levelsincreased in SFA-HFD mice compared with MUFA-HFDmice, while plasma triacylglycerol, nonesterified fatty acid,cholesterol, IL-1b, and IL-6 were equivalent (Supplemen-tary Table 2).

MUFA-HFD Mice Exhibit a Hyperplastic AdiposePhenotype, With Enhanced AMPK ActivationCompared With SFA-HFD Mice

MUFA-HFD mice displayed adipose hyperplasia comparedwith SFA-HFD mice (Fig. 3A and B). The expression of adipo-genic markers PPARg and PGC-1a were increased in MUFAadipocytes (Fig. 3D). We demonstrate that preadipocyte-enriched MUFA SVF exhibited greater PGC-1a and Forkheadbox protein class O1 (FOXO-1) mRNA expression in re-sponse to adipogenic media compared with SFA SVF (Fig.3E). Previous studies demonstrated that AMPK activa-tion regulates the NLRP3 inflammasome complex (14)

Figure 1—HFDs enriched with PA can prime pro-IL-1b production in the SVF of adipose tissue in vivo. A: Adipose tissue from mice feda chow diet, a MUFA-HFD, or an SFA-HFD for 16 weeks was cultured in complete media (100 mg/mL) for 24 h, and levels of IL-1b secretedinto culture media were measured by ELISA (n = 5–6). B: Protein levels of mature ASC, procaspase-1, active IL-1b, and control GAPDH inadipose tissue were determined by immunoblot analysis. Protein bands were quantified by densitometry and normalized to GAPDH levels(n = 7–8). C: Gene expression analysis of SIRT-1, TLR4, NLRP3, IL-18, IL-1b, and caspase-1 in adipose tissue (n = 6–8, normalized to chowcontrol) was quantified by real-time PCR. D: Adipocytes and SVFs were isolated from epididymal fat pads by collagenase digestion. IL-1bsecretion from the SVF (1 3 106 cells/mL) and adipocytes (200 mL packed volume/mL) cultured in complete media for 24 h with or withoutATP (5 mmol/L) was determined by ELISA (n = 7–8). E: The SVF was seeded at a density of 1 3 106 cells/mL and cultured for 24 h with orwithout ATP (5 mmol/L). Caspase-1 activity was determined. F: Protein expression of pro-IL-1b, mature IL-1b, and control GAPDH in theSVF treated with LPS (10 ng/mL) for 3 h was determined by immunoblot analysis. A representative immunoblot is shown. Protein bandswere quantified by densitometry and normalized to GAPDH levels (n = 7–8). *P < 0.05, **P < 0.01, ***P < 0.001, chow-fed vs. HFD-fedmice; #P < 0.05, ##P < 0.01, ###P < 0.001 MUFA vs. SFA for all graphs. White bars, chow-fed mice; striped bar, MUFA-HFD–fed mice;black bars, SFA-HFD–fed mice in all graphs.

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and that MUFA can enhance AMPK activity (15). We thusspeculated that differential AMPK activity after HFDs mayaccount for alterations in adipose IL-1b levels. Equivalentlevels of pAMPK were observed in adipose tissue fromchow-fed and MUFA-HFD–fed mice, but levels were signif-icantly reduced after the SFA-HFD (Fig. 3F).

OA Impedes ATP-Induced Secretion of IL-1b in LPS-Primed BMMs in an AMPK-Dependent MannerBMMs mimic the IL-1–related immuno-phenotype of adi-pose tissue macrophages, and we thus used them in

mechanistic studies (16). MUFA-HFD–derived BMMs se-creted significantly less IL-1b after LPS and ATP stimulationcompared with SFA-derived BMMs ex vivo (Fig. 4A).Caspase-1 activity was significantly higher in BMMs fromSFA-HFD–fed mice compared with BMMs from chow-fedand MUFA-HFD–fed mice (Fig. 4B). Pretreatment of LPS-primed BMMs with an NLRP3 inflammasome inhibitor,CRID3, attenuated IL-1b secretion from BMMs from SFA-HFD–fed mice (Fig. 4C). Furthermore, in LPS-primed BMMs,PA, but not OA, increased pro-IL-1b secretion, and inducedIL-1b (Fig. 4D–F) and IL-18 secretion (Supplementary Fig. 5D).

Figure 2—A MUFA-enriched HFD partially improves insulin sensitivity and hyperinsulinemia. ITT (0.5 units/kg insulin) (A) and GTT (1.5 g/kgglucose) (B) results are shown in 6-h–fasted chow-fed, MUFA-HFD–fed, and SFA-HFD–fed animals (n = 12–14). C: Insulin secretionresponse in overnight-fasted mice after intraperitoneal injection with 1.5 g/kg glucose. Tail-vein blood samples were taken at indicatedtimes, and insulin levels were determined by ELISA (n = 7–12). In A–C: Black circles, chow-fed mice; white squares, MUFA-HFD–fed mice;black squares, SFA-HFD–fed mice. D: Gene expression analysis of GLUT-4 and IRS-1 in primary adipocytes from mice fed chow, a MUFA-HFD, and an SFA-HFD, as measured by real-time PCR (n = 5). E: Levels of tyrosine pIRS-1 in adipose tissue protein lysates were measuredusing a PathScan ELISA kit. The fold increase in response to insulin over basal (non-insulin stimulated) in adipose tissue for each individualmouse was calculated and is presented (n = 4–8). F: Adipose tissue explants (50 mg) were stimulated with insulin (100 nmol/L) ex vivo for15 min, and protein lysates were prepared. Phosphorylated AKT, whole-cell AKT, and control GAPDH levels were determined by immu-noblot analysis. A representative immunoblot is shown. Protein bands were quantified by densitometry and normalized to GAPDH levels(n = 6). In D–F: White bar, chow-fed mice; striped bar, MUFA-HFD–fed mice; black bar, SFA-HFD–fed mice. *P < 0.05, **P < 0.01, ***P <0.001, chow-fed vs. HFD-fed mice ; #P < 0.05, ###P < 0.001, MUFA vs. SFA for all graphs.

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We speculated that MUFA-induced AMPK may impedeNLRP3 inflammasome activity. Interestingly, OA pre-vented ATP-induced IL-1b secretion in LPS-primedBMMs (Fig. 4D). Furthermore, PA-treated BMMs ex-hibited lower pAMPK levels compared with OA-treatedcells, consistent with findings in adipose tissue (Fig. 4Eand F). Pretreatment of LPS-primed BMMs with anAMPK inhibitor, compound C, prior to OA treatment re-duced pAMPK (Supplementary Fig. 5E) and increased IL-1band IL-18 secretion (Fig. 5A and Supplementary Fig.5F). Conversely, pretreatment with the AMPK agonistAICAR completely reverses PA-induced IL-1b secretion(Fig. 5B). We similarly demonstrated that OA can preventATP-stimulated IL-1b secretion in LPS- and PA-primedmacrophages (Fig. 5C). These findings demonstrate thatMUFAs not only fail to prime IL-1b, but can also blockATP-induced IL-1b secretion in preprimed cells in anAMPK-dependent manner. To confirm, stable THP-1 cell

lines lacking the AMPKa1 subunit were created. Phorbolmyristic acid (PMA)–differentiated THP-1 macrophagessecrete high levels of IL-1b in response to PA, but notto OA, similar to BMMs (Fig. 5D). Supporting our chem-ical inhibitor studies, knockdown of AMPKa1 increasedIL-1b secretion from OA-treated cells in the presence andabsence of ATP (Fig. 5E and F).

Regression Study: Replacing SFA-HFD With MUFA-HFD Cannot Reverse Adipose Dysfunction or IRMice were fed an SFA-HFD for 16 weeks to induce anobese and insulin-resistant state, and were subsequentlymaintained on an SFA-HFD or switched to a MUFA-HFDto establish whether we could regress established IR.Switching to a MUFA-HFD only moderately improvedHOMA-IR, but it did not reach statistical significance (Fig.6A), whereas ITT (Fig. 6B) and GTT curves were un-changed (data not shown). Nonetheless, switching from

Figure 3—MUFA-HFD–fed mice exhibit hyperplastic adipose morphology, with enhanced AMPK activation. A and B: Adipocyte size wasmonitored in paraffin-embedded adipose tissue samples by hematoxylin-eosin staining (n = 4–5). C: Weight of epididymal adipose tissue(EAT) and visceral adipose tissue (VAT) and subcutaneous (SC), perirenal (PR), and brown (BAT) adipose tissue depots of chow-fed, MUFA-HFD–fed, and SFA-HFD–fed mice (n = 10–18). D: Primary adipocytes were harvested from adipose tissue by collagenase digestion, andadipocyte mRNA of PPARg and PGC-1a was measured by real-time PCR (n = 5–6). E: SVF was cultured for 7 days and then exposed toadipocyte-differentiating media (DM) for 24 h. PGC-1a and FOXO-1 mRNA expression was measured by RT-PCR (n = 8–12). Results areexpressed as the fold change relative to SVF cultured in the absence of DM. F: Adipose tissue protein expression of pAMPK, whole-cellAMPK, and GAPDH was determined by immunoblot analysis. A representative immunoblot is shown. Protein bands were quantified bydensitometry and normalized to GAPDH levels (n = 7–9). White bar, chow-fed mice; striped bar, MUFA-HFD–fed mice; black bar, SFA-HFD–fed mice for all graphs. *P< 0.05, **P< 0.01, ***P< 0.001, chow-fed vs. HFD-fed mice; #P< 0.05, ##P< 0.01, ###P< 0.001, MUFAvs. SFA for all graphs.

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an SFA-HFD to a MUFA-HFD prevented further increasesin fasting plasma insulin concentrations (Fig. 6C) and in-sulin secretion in response to glucose (Fig. 6D). Adiposemorphology was equally hypertrophic between groups(Fig. 6E). Adipose M1 macrophage infiltration was alsoequivalent (data not shown). Pancreatic cell size was

reduced after a MUFA-HFD compared with an SFA-HFD(Fig. 6F).

Habitual Dietary MUFA Is Associated With EnhancedInsulin Sensitivity in Human CohortTo translate our findings from animal to humans, wereanalyzed data from two human studies with detailed

Figure 4—OA impedes ATP-induced secretion of IL-1b. A: BMMs from chow-fed, MUFA-HFD–fed, and SFA-HFD–fed mice were isolatedand were stimulated with or without LPS (10 ng/mL) for 3 h, followed by ATP (5 mmol/L) stimulation for 1 h. IL-1b secretion into culturemedia was measured by ELISA. White bars, control; black bars, +LPS+ATP. ***P < 0.001, control vs. +LPS+ATP within each experimentalgroup; ###P < 0.001, MUFA-HFD–fed vs. SFA-HFD–fed mice; n = 3. B: Basal levels of caspase-1 activity in BMMs derived from chow-fed,MUFA-HFD–fed, and SFA-HFD–fed mice were measured by ELISA. White bar, chow-fed mice; striped bar, MUFA-fed mice; black bar,SFA-fed mice. #P< 0.05, MUFA vs. SFA; n = 3. C: BMMs derived from chow-fed, MUFA-HFD–fed, and SFA-HFD–fed mice were seeded ata density of 13 106 cells/mL. BMMs were primed with LPS (10 ng/mL) for 6 h, pretreated with vehicle control (DMSO) or CRID3 (50 nmol/L)for 30 min and stimulated with or without ATP (5 mmol/L) for 1 h. IL-1b secretion into culture media was measured (white bar, untreated;striped bar, LPS+DMSO+ATP; black bar, LPS+CRID3+ATP). ***P < 0.001, w.r.t. SFA+CRID3; n = 6–8. D: BMMs were harvested from leanmice and seeded at a density of 1 3 106 cells/mL. Cells were treated with LPS (10 ng/mL) for 3 h, washed with PBS prior to the addition offresh media, and then stimulated with PA (250 mmol/L) or OA (250 mmol/L) for 24 h. BMMs were subsequently stimulated with or withoutATP (5 mmol/L) for 1 h. Media and protein lysates were harvested. IL-1b secretion into culture media was measured by ELISA (###P <0.001; ***P < 0.001, LPS+PA+ATP vs. LPS+OA+ATP; n = 4). E: Densitometry analysis of pro-IL-1b, pAMPK, AMPK, and GAPDH in fattyacid–treated BMMs (*P< 0.05, LPS+PA vs. LPS+OA; *P < 0.05, LPS+PA+ATP vs. LPS+OA+ATP; n = 4). F: Representative blot of pro-IL-1b,pAMPK, AMPK, and GAPDH protein levels in fatty acid–treated BMMs. FA, fatty acid.

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dietary fat data wherein we could discriminate betweenfat quantity and composition. In T2D subjects(CORDIOPREV cohort), high habitual SFA intake, butnot MUFA intake, was associated with significantly lowerinsulin sensitivity, as measured by ISI (Fig. 7A) andHOMA-IR (Fig. 7B). Further, hs-CRP increased withhigher habitual SFA intake (Fig. 7C). Age, BMI, waist cir-cumference, and systolic blood pressure were not differentbetween dietary SFA tertiles (Supplementary Table 3).

Second, a subcohort of subjects with metabolic syndromefrom the LIPGENE study was categorized according totheir habitual dietary fat intake. At baseline, individualswith habitual high SFA intake (Supplementary Fig. 6A)exhibited elevated caspase-1 (P = 0.005) and pycard-1(P = 0.009) mRNA expression in adipose tissue (Supple-mentary Fig. 6B and C). Dietary intervention with MUFAfor 16 weeks resulted in improvements in the acute in-sulin response to glucose and the first-phase insulin

Figure 5—OA modulates IL-1b in an AMPK-dependent manner. BMMs from chow-fed mice were primed with LPS (10 ng/mL) for 3 h,pretreated with AMPK inhibitor compound C (CC) (10 mmol/L) or vehicle control (DMSO) for 1 h, exposed to OA (250 mmol/L) for 24 h, andstimulated with ATP (5 mmol/L) for 1 h. A: IL-1b secretion into culture media was measured by ELISA (*P< 0.05, OA-CC vs. OA+CC; n = 4).B: BMMs pretreated with or without AICAR (100 mmol/L) for 30 min, then exposed to PA (250 mmol/L) or OA (250 mmol/L) for 24 h andstimulated with ATP (5 mmol/L) for 1 h. IL-1b secretion into culture media was measured by ELISA (***P < 0.001 PA vs. AICAR+PA; n = 6).C: LPS-primed BMMs pretreated with or without OA (250 mmol/L) for 1 h, then exposed to PA (250 mmol/L) for 24 h and stimulated with ATP(5 mmol/L) for 1 h. IL-1b secretion into culture media was measured by ELISA (***P < 0.001, PA vs. PA pretreated with OA; n = 4–8).D: THP-1 human monocytes differentiated to macrophages by culturing in the presence of PMA (100 ng/mL) for 24 h. Media were removed,and fresh media were added overnight. THP-1 macrophages were treated with PA (200 mmol/L) or OA (200 mmol/L) with or without ATP(5 mmol/L) for 4 h. IL-1b secretion into culture media was measured by ELISA (***P < 0.001, PA vs. OA; n = 4). E: Immunoblot demonstratingsuccessful lentiviral shRNA-mediated knockdown of AMPKa1 subunit in THP-1 monocytes. F: THP-1 shAMPK or control knockdown celllines were differentiated from macrophages (100 ng/mL PMA) for 24 h. Media were removed, and fresh media were added overnight. Cellswere treated with OA (200 mmol/L) with or without ATP (5 mmol/L) for 4 h. IL-1b secretion into culture media was measured by ELISA(expressed as fold change relative to UT). AU, arbitrary units; FA, fatty acid; UT, untreated.

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response (Supplementary Fig. 6D and E) in habitualhigh-MUFA consumers, but not in high-SFA consumers.Age, preintervention HOMA-IR, and habitual MUFA intakewere the most important determinants of improved acuteinsulin response to glucose in response to a MUFA-HFD(r2 = 0.620, P = 0.015) (Supplementary Table 4).

DISCUSSION

This study has demonstrated that enrichment of obesi-genic HFDs with MUFA can improve insulin sensitivity,reduce adipose IL-1b–mediated inflammation, and pro-mote adipose hyperplasia compared with diets enriched

with SFA. We further demonstrate that MUFA-HFDs failto prime IL-1b in whole adipose tissue and the SVF ofadipose tissue, with reductions in both active IL-1b pro-tein levels and ATP-induced IL-1b secretion. Lack of IL-1bcoincided with the preservation of adipose AMPK activityin the MUFA group, which has previously been implicatedin impeding NLRP3 inflammasome activity (5). Wefurther demonstrate that the MUFA OA can preventATP-induced IL-1b secretion in both LPS- and PA-primedmacrophages in vitro, indicative that MUFA can impedeNLRP3 inflammasome activation. Furthermore, pretreat-ment of cells with an AMPK inhibitor rescued ATP-induced

Figure 6— Regression study: replacing SFA-HFD with a MUFA-HFD cannot reverse IR. Mice were fed an SFA-HFD for 16 weeks and weresubsequently maintained on an SFA-HFD or were switched to a MUFA-HFD for a further 16 weeks. A: HOMA-IR was calculated based onfasting glucose and insulin concentrations. Black bars, SFA; white bars, SFA → MUFA; n = 10. B: ITT (0.5 units/kg insulin) results in 4- to6-h–fasted animals (n = 10). C: Fasting plasma insulin levels over time (n = 10). D: Insulin secretion response to glucose challenge (1.5 g/kg)in overnight-fasted mice (n = 10). In B–D: Black squares, SFA-HFA; white squares, SFA → MUFA. E and F: Adipocyte and pancreatic cellsizes were monitored in paraffin-embedded tissue samples by hematoxylin-eosin staining in SFA and SFA → MUFA mice (black bar, SFA;white bar, SFA → MUFA; n = 3–4). #P < 0.05, ##P < 0.01, w.r.t. SFA → MUFA in all graphs.

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IL-1b secretion in the presence of OA. These findingsindicate that MUFA, via activation of AMPK, can impedeATP-induced processing of pro-IL-1b to active IL-1b.Indeed, preservation of adipose AMPK activity in

MUFA-HFD–fed mice may account for reduced IL-1bin adipose tissue and improved insulin sensitivity invivo. In regression studies, the replacement of SFA forMUFA did not significantly rescue IR, which is associated

Figure 7—Habitual dietary SFA, but not MUFA, promotes IR. A–C: T2D subjects from the CORDIOPREV study (NCT00924937) were categorizedbased on tertiles of baseline fasting plasma SFA and MUFA concentrations. A: ISI is represented according to SFA and MUFA tertiles. B: HOMA-IRis represented according to SFA and MUFA tertiles. C: C-reactive protein is represented according to SFA tertiles. Black bars, SFA. In A–C: **P <0.01, w.r.t. tertile 1; #P < 0.05, w.r.t tertile 2; n = 160–184. D: Schematic representation illustrating the differential effects of an SFA-HFD vs.a MUFA-HFD on pro-IL-1b priming and NLRP3 inflammasome activation: 1, MUFA-HFD lacks the ability to prime pro-IL-1b in SVF; 2, MUFA-HFDmaintains adipose protein pAMPK levels at those of chow-fed mice, while SFA-HFD–fed mice display reduced pAMPK levels; 3, caspase-1 activityis significantly increased in BMMs from SFA-HFD–fed mice; 4, greater levels of IL-1b are secreted from SFA SVF compared with MUFA SVF; 5,protein pAKT levels are reduced in SFA adipose tissue compared with MUFA adipose tissue; and 6, a MUFA-HFD induced a hyperplastic adiposemorphology, while an SFA-HFD induced adipocyte hypertrophy. NFkB, nuclear factor-kB; ROS, reactive oxygen species.

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with IL-1b activation and hypertrophic adipose morphol-ogy, but did lower fasting plasma insulin levels at baselineand after a glucose challenge. Human studies highlight theimportance of habitual dietary fat composition, suggestingthat high habitual MUFA intake is associated with increasedinsulin sensitivity and greater flexibility in responding todifferent dietary interventions.

It is well acknowledged that IL-1b, which is primed bySFA-HFD and cleaved via the NLRP3 inflammasome com-plex, has a detrimental role in obesity-induced IR (3,5,14).Our work demonstrates that MUFA-HFD–induced obesitydid not induce adipose IL-1b production, coincident withlower caspase-1 activity, and reduced NLRP3, caspase-1,IL-1b, and IL-1R1 mRNA expression in the SVF. Thesefindings extend those of Wen et al. (5), who demonstratedthat treatment of BMMs with OA did not enhance LPS-induced IL-1b ex vivo. Similarly, we show that OA doesnot prime IL-1b in vitro. Furthermore, we establish thatthe treatment of LPS-primed macrophages with OA, priorto an ATP stimulus, markedly attenuates IL-1b secretionfrom macrophages. These findings reveal that not only doMUFAs fail to prime IL-1b, but they also impede NLRP3-mediated processing of IL-1b in preprimed cells.

Lack of IL-1b signaling is accompanied by improvedinsulin sensitivity in HFD-fed mice (6). In our study,MUFA-HFD mice have improved insulin sensitivity.Markers of inflammation and insulin sensitivity are com-parable in periphery tissues between HFD groups, sug-gesting the modulation of adipose IL-1b by MUFA iscritical to the insulin-sensitive phenotype. Interestingly,MUFA-HFD fails to improve glucose tolerance. We spec-ulated that altered hepatic glucose production may ac-count for this phenotype; however, hepatic G-6-P andPepCK2 expression were equivalent, while basal glucoselevels were reduced with MUFA-HFD. MUFA-HFD–fedmice secrete significantly lower levels of insulin in re-sponse to a glucose load and have reduced pancreaticcell death compared with SFA-HFD–fed mice. Addition-ally, by using a regression dietary model we display evi-dence that switching from an SFA-HFD to a MUFA-HFDhalted the progression of HFD-induced pancreatic hyper-trophy and hyperinsulinemia. Maedler et al. (17) demon-strated that MUFA, both C16:1 and C18:1, prevented thedeleterious effects of palmitate on pancreatic b-cell func-tion in vitro. We postulate that the pancreatic cells maybe partially protected by mice being fed a MUFA-HFD invivo, and as a result insulin secretion levels are not ele-vated to the same extent as those associated with anSFA-HFD, which may account for the lack of differencein glucose tolerance.

Hypertrophied adipose tissue is associated with adi-pose dysfunction, lipid spillover, and ectopic hepaticdeposition (18). Hyperplastic adipose tissue is associatedwith insulin sensitivity independent of body fat in humans(19). Artificial induction of adipogenesis in the subcuta-neous adipose tissue of obese mice improves glucose tol-erance and HOMA-IR (20). In our study, adipose tissue

from MUFA-HFD–fed mice displays a hyperplastic pheno-type with reduced M1 adipose tissue macrophages andimproved insulin sensitivity. Adipocyte PPARg and PGC-1a were markedly increased after mice were fed theMUFA-HFD. Furthermore, PGC-1a and FOXO-1 levelswere amplified in MUFA-HFD SVF compared with SFA-HFD SVF after ex vivo stimulation with adipogenic media.Together, these results are indicative of an increased adi-pogenic potential of preadipocytes in MUFA-HFD–fedmice. It is well established that IL-1b impedes adipogen-esis in 3T3-L1 adipocytes (6). We propose that the hyper-plastic adipose morphology may be due to attenuatedIL-1b within the microenvironment of adipose tissuefrom MUFA-HFD–fed mice.

AMPK is a pleiotropic metabolic sensor (21), whichalso modulates inflammation (22). Inhibition of AMPKby SFA attenuates autophagy and increases mitochondrialdysfunction via reactive oxygen species production, whichin turn activate the NLRP3 inflammasome (5,23). MUFAhas been shown to activate AMPK in aortic tissue (15).Moreover, oleate prevents palmitate-induced ER stress inskeletal muscle in an AMPK-dependent manner, contrib-uting to improved local insulin sensitivity (24). However,it has not been shown whether OA regulates AMPK inadipose tissue and in turn modulates IL-1b release. Ourwork has demonstrated that pAMPK levels are equivalentin both chow-fed and MUFA-HFD–fed mouse adipose tis-sue, and activation is significantly reduced after mice werefed an SFA-HFD. This reduction in pAMPK is associatedwith the upregulation of mature IL-1b levels in SFA-HFD–fed mouse adipose tissue. We thus postulated thatMUFA may signal via the AMPK pathway to disruptNLRP3 inflammasome activation. In support of our hy-pothesis, we demonstrate that treatment of LPS-primedmacrophages with an AMPK inhibitor can rescue ATP-induced IL-1b secretion in the presence of OA. AMPKa1knockdown experiments corroborate this finding, exhibit-ing increased IL-1b secretion in the presence of OA. Thesenovel findings suggest that the beneficial effects ofa MUFA-HFD may be mediated via preservation ofAMPK activity and reduced processing of IL-1b in theobese state. A recent study (14) illustrated that metfor-min attenuates IL-1bmaturation in T2D patients throughAMPK activation, which is akin to the MUFA-HFD andOA pretreatment.

However, we cannot overlook the effect of AMPKactivation in relation to energy homeostasis. ObeseMUFA-HFD–fed animals exhibited greater energy expen-diture, with increased VO2 and heat production. This mayalso be attributable to AMPK activation, which is knownto increase fatty acid oxidation and promote mitochon-drial biogenesis. Recently, Freigang et al. (25) demon-strated that OA-mediated mitochondrial uncouplinginhibited ATP-induced IL-1b production. However, inthis study mRNA markers of mitochondrial biogenesiswere comparable between MUFA-HFD–fed and SFA-HFD–fed groups in adipose, liver, and skeletal muscle

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tissue. Nevertheless, the difference in adipose morphol-ogy, IL-1b levels, and IR remain true when weightmatched.

Given the beneficial effects of MUFA on inflammationand insulin sensitivity observed in our animal andcellular models, we examined whether habitual dietaryMUFA intake was associated with insulin sensitivity andreduced inflammation in humans. In T2D subjects(CORDIOPREV cohort), high habitual SFA intake, butnot high MUFA intake, was associated with IR. Further-more, individuals with metabolic syndrome (LIPGENEcohort) with high habitual MUFA intake at baselineexhibited lower adipose tissue caspase-1 and pycard-1mRNA levels, and demonstrated increased insulin sensi-tivity after a MUFA dietary intervention compared withhabitual high SFA consumers. These studies suggest thatindividuals with high habitual MUFA intake exhibita certain degree of flexibility in response to dietaryinterventions, which was not evident in individuals withhabitual high SFA intake.

In summary, we provide the first in vivo evidence thatobesigenic diets enriched with MUFA can lower adiposeIL-1b secretion, stimulate adipose hyperplasia, and re-duce IR that is ordinarily associated with SFA-inducedobesity (Fig. 7D). We further demonstrate in vitro thatMUFA can block ATP-induced processing of IL-1b inpreprimed cells, in an AMPK-dependent manner. Thesefindings indicate that MUFAs may limit SFA-inducedIL-1b levels and associated adverse metabolic sequelaein the obese state.

Acknowledgments. The authors thank the staff of the core-technologiesin the Conway Institute of Biomedical and Biomolecular Research and Univer-sity College Dublin (UCD) Biomedical Facility (Belfield, Dublin) for technical sup-port. The authors also thank Dr. Daniel Jones, Conway Institute of Biomedicaland Biomolecular Research, UCD, for generating our histological data; andDr. Eugene Dempsey, Conway Institute of Biomedical and Biomolecular Re-search, UCD, for the THP-1 cells. In addition, the authors thank the subjectsand investigators of the CORDIOPREV and LIPGENE studies.Funding. The work presented in this article has been supported by ScienceFoundation Ireland (grant SFI PI/11/1119). The CORDIOPREV and LIPGENE studysubjects and investigators were funded by European Commission FP6 (grantFOOD-CT-2003-505944).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. O.M.F. researched the data and cowrote themanuscript. C.L.L., A.M.M., and C.M.R. researched the data and proofread themanuscript. R.K. generated the stable AMPKa knockdown cell line. N.P.H., A.A.C.,and M.E.O. researched the data. R.C.C. facilitated the CRID3 experiments. L.M.and K.N.N. performed the energy expenditure analysis. A.C.T. and M.J.M.performed the LIPGENE reanalysis. J.F.A.-D. and J.L.-M. participated in theCórdoba study and reviewed the data. D.P.O. provided the vector constructs.L.A.O. facilitated the CRID3 experiments and reviewed the data. F.C.M. designedthe study, reviewed the data, and edited the manuscript. H.M.R. designed thestudy, reviewed the data, and cowrote the manuscript. All authors have approvedthe final version of the manuscript. H.M.R. is the guarantor of this work and, as

such, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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