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Omega-3 Fatty Acids Reduce Adipose TissueMacrophages in Human Subjects With Insulin ResistanceMichael Spencer,1Brian S.Finlin,1Resat Unal,1Beibei Zhu,1Andrew J.Morris,2Lindsey R.Shipp,1Jonah Lee,3R.Grace Walton,1Akosua Adu,1Rod Erfani,3Marilyn Campbell,3Robert E.McGehee Jr.,4Charlotte A.Peterson,3and Philip A.Kern1Fish oils(FOs)have anti-inflammatory effects and lower serumtriglycerides.This study examined adipose and muscle inflam-matory markers after treatment of humans with FOs andmeasured the effects of v-3 fatty acids on adipocytes and macro-phages in vitro.Insulin-resistant,nondiabetic subjects weretreated with Omega-3-Acid Ethyl Esters(4 g/day)or placebo for12 weeks.Plasma macrophage chemoattractant protein 1(MCP-1)levels were reduced by FO,but the levels of other cytokines wereunchanged.The adipose(but not muscle)of FO-treated subjectsdemonstrated a decrease in macrophages,a decrease in MCP-1,and an increase in capillaries,and subjects with the most macro-phages demonstrated the greatest response to treatment.Adiposeand muscle v-3 fatty acid content increased after treatment;how-ever,there was no change in insulin sensitivity or adiponectin.Invitro,M1-polarized macrophages expressed high levels of MCP-1.The addition of v-3 fatty acids reduced MCP-1 expression withno effect on TNF-a.In addition,v-3 fatty acids suppressed theupregulation of adipocyte MCP-1 that occurred when adipocyteswere cocultured with macrophages.Thus,FO reduced adiposemacrophages,increased capillaries,and reduced MCP-1 expres-sion in insulin-resistant humans and in macrophages and adipo-cytes in vitro;however,there was no measureable effect oninsulin sensitivity.Diabetes 62:17091717,2013The development of type 2 diabetes representsa complex series of events that begins with thedevelopment of insulin resistance.The changesin adipose tissue that accompany obesity,themetabolic syndrome,and insulin resistance include in-creased adipose tissue macrophages,circulating inflam-matory markers such as tumor necrosis factor-a(TNF-a)and interleukin(IL)-6(13),and the development ofa chronic inflammatory state.In addition to the infiltrationof macrophages,other changes occur in the adipose tissueof obese,insulin-resistant subjects,including an increasein extracellular matrix(ECM)components,such as colla-gen VI,thrombospondin,and collagen V and a decrease inelastin(47).Along with adipocyte expansion,changes inthe adipose vasculature have been described,includinga decrease in capillaries and an increase in larger bloodvessels(7,8),leading to the hypothesis that adipocyte ne-crosis and inflammation develop as a result of adipocyteexpansion into a relatively hypoxic,nonelastic ECM(9).Fish oils(FOs)are rich sources of v-3 polyunsaturatedfatty acids(v-3 PUFAs),and there is a large amount ofliterature on the potential benefits of FOs on loweringserum triglycerides,cardiovascular protection,and im-mune modulation.There is considerable evidence sup-porting the anti-inflammatory effects of v-3 PUFAs(10),and FOs may be an adjunct in the treatment of rheumatoidarthritis,inflammatory bowel disease,and asthma(11,12).Although the mechanism of this effect is complex,partof the anti-inflammatory action involves an inhibition ofthe production of eicosanoids from arachadonic acid(13).In addition,a number of studies have demonstrated thatFOs have a peroxisome proliferatoractivated receptor g(PPARg)like effect(14).PPARg agonist drugs,such asthe thiazolidinediones,improve insulin sensitivity and haveanti-inflammatory properties.Previous studies have dem-onstrated thiazolidinedione-mediated reductions in plasmainflammatory markers and adipose tissue macrophagesand an increase in blood adiponectin(1518).Although theeffects of FOs on adipose inflammation are unknown,previous studies have generally not found that FOs im-prove insulin sensitivity in humans(19).This study was performed to determine whether FOswould ameliorate the adipose tissue inflammation,fibrosis,and vascular abnormalities that are found in subjects withobesity and insulin resistance.After 12 weeks of treatmentwith standard clinical doses of v-3 PUFAs,we founda decrease in adipose tissue macrophages,an increase inadipose capillaries,and a decrease in macrophage che-moattractant protein 1(MCP-1)levels.RESEARCH DESIGN AND METHODSHuman subjects.Nondiabeticsubjectswith eitherimpaired glucose tolerance,impaired fasting glucose,or at least three features of the metabolic syndromewere recruited.The participants signed consent forms that were approved bythe institutional review boards from either the University of Arkansas forMedical Sciences or the University of Kentucky.Participants were excluded forany history of coronary disease,history of inflammatory disease,or the chronicuse of any anti-inflammatory medication or other medication likely to changeadipocyte metabolism.No subjects were consuming v-3 PUFA supplements orexcessive quantities of foods containing v-3 PUFAs.A baseline food historyquestionnaire was administered,and no subject was consuming.0.7 g/day oftotal v-3 PUFAs,and,2%of the v-3 PUFAs were from marine sources.Baseline measures included oral glucose tolerance test,serum lipids,thyroidfunction,and routine laboratories(liver enzymes,creatinine,and electrolytes)to exclude type 2 diabetes or other chronic conditions.If subjects met theinclusion criteria,insulin sensitivity was measured with a frequently sampledintravenous glucose tolerance test,as described previously(7,20,21).This testyields a robust index of insulin sensitivity(SI)that correlates well with theglucose disposal rate from the euglycemic clamp(22).All participants un-derwent an incisional abdominal adipose biopsy in order to remove;4 g oftissue and a needle muscle biopsy.Subjects were then randomized to receiveFrom the1Department of Medicine,Division of Endocrinology,and the Barn-stable Brown Diabetes and Obesity Center,University of Kentucky,Lexing-ton,Kentucky;the2Division of Cardiovascular Medicine,University ofKentucky,Lexington,Kentucky;the3College of Health Sciences,Universityof Kentucky,Lexington,Kentucky;and the4Department of Pediatrics,Uni-versity of Arkansas for Medical Sciences,Little Rock,Arkansas.Corresponding author:Philip A.Kern,philipkernuky.edu.Received 2 August 2012 and accepted 23 November 2012.DOI:10.2337/db12-1042.Clinical trial reg.no.NCT00579436,clinicaltrials.gov.This article contains Supplementary Data online at http:/diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1042/-/DC1.?2013 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,and the work is not altered.See http:/creativecommons.org/licenses/by-nc-nd/3.0/for details.diabetes.diabetesjournals.orgDIABETES,VOL.62,MAY 20131709ORIGINAL ARTICLEeither Omega-3-Acid Ethyl Esters(LOVAZA,FO),4 g/day,or identicallypackaged placebo(corn oil).FO and matching placebo tablets were suppliedby GlaxoSmithKline(Research Triangle Park,NC).The subjects were un-aware of their drug/placebo assignment and reported no gastrointestinal sideeffects or“fish taste.”Of the 34 participants,23 had either impaired glucose tolerance or impairedfasting glucose and 22 were women.Table 1 shows the clinical features of thesubjects that were randomized into either the FO or placebo group.Therewere no significant differences between the groups in BMI,age,sex,SI,lipids,or glucose.The mean BMI was 33 kg/m2in both groups(range,2743).Hypertriglyceridemia was not a criteria for selection into the study,and meanfasting triglycerides were 163 6 23 and 150 6 7.6 mg/dL in the placebo and FOgroups,respectively.Plasma cytokines.Plasma cytokines were measured using Luminex assays,using the Milliplex cytokine high sensitivity assay,the human metabolic hor-mone assay,and adipokine A assay kits.Total and high-molecular-weightadiponectin were measured using a multimeric adiponectin ELISA kit(Alpco,Salem,NH).Histochemistry and immunohistochemistry.As described previously(5),adipose samples were fixed in Bouins solution,paraffin embedded,and cutinto 5-mm-thick sections.After a series of xylene/alcohol washes,the slideswere stained,dehydrated,and mounted in cytoseal(Richard-Allan Scientific,Kalamazoo,MI).To assess fibrosis in adipose tissue,a Massons trichromestain(Trichrome Stain Kit,HT15;Sigma-Aldrich)was used,along with stainingfor collagen VI(rabbit biotinylated anti-human ColVI,C7510-61Z;US Bi-ological)(5).Quantification of fibrosis was performed as previously described(7),using National Institutes of Health ImageJ on the composite photographedimages to identify the stained areas in relation to the entire area.Macrophageswere identified in the adipose tissue using an antibody to CD68(clone KP1;Dako)(5)and visualized using a horseradish peroxidaseconjugated second-ary antibody with color development using a diaminobenzidine(DAB)sub-strate.Ten random fields were photographed for each sample using the 103objective,and a macrophage counting routine was developed in ImageJ soft-ware to avoid subjectivity issues that can be introduced by manual counting.The counting routine was verified by three independent manual counts ona subset of slides under blinded conditions where the subject identity andtreatment were not known(5).The quantitation of blood vessels was performed by performing doublestaining for endothelial cells,using a biotin-labeled Ulex europaeus agglutinin(UEA)lectin(L8262;Sigma-Aldrich)and a monoclonal antibody to alphasmooth muscle actin(ASMA)(SC130616;Santa Cruz Biotech).Capillariesstained only with the endothelial stain,whereas larger blood vessels wereidentified by the ASMA ring around the endothelial cells,as described previ-ously(7).The antigens were stained using a mixture of UEA lectin(40 mg/mL)and ASMA antibody(1:25 dilution)diluted in 2.5%horse serum(S-2012;VectorLaboratories).Each antigen was visualized in a sequential manner.UEA wasdeveloped first with streptavidin-conjugated horseradish peroxidase enzyme(SA-5004;Vector Laboratories)followed by color development using theImmPACT NovaRED peroxidase substrate kit(SK-4805;Vector Laboratories).ASMA-bound antibody was identified with the ImmPRESS anti-mouse Ig(peroxidase)polymer detection kit(MP-7402;Vector Laboratories)and visu-alized using the ImmPACT SG peroxidase(SK-4705;Vector Laboratories).These dyes exhibit different spectral peaks,allowing for easy separation.Twenty random fields were photographed for each subject using the 203objective with the Nuance multispectral camera(Caliper Life Sciences,Hopkinton,MA).This system captures a photograph of the sample at 10-nmintervals from 435 to 760 nm to build a spectral graph of the sample andallowing for separation of the spectra of each chromogenic dye.Capillariesand vessels were manually counted under blinded conditions,as describedabove.The number of capillaries and larger vessels were normalized totissue surface area as measured by ImageJ software.RNA isolation and mRNA analysis.Total RNA was isolated from humanadipose tissue using RNAeasy Lipid Tissue Mini(Qiagen,Valencia,CA),andRNA quantity and quality were verified using an Agilent 2100 Bioanalyzer(PaloAlto,CA).Real-time RT-PCR was performed as described previously(23),where 18SRNA was used as a standard to control for differences in individual samples.The primers used for MCP-1,CD68,and 18S were described previously(23).To obtain high-throughput measures of gene expression in response to FOtreatment,we used the NanoString nCounter analysis system(NanoStringTechnologies,Seattle,WA)on 36 samples,including pre-and posttreatmentsamples from nine placebo-treated subjects and nine FO-treated subjects.NanoString allows the multiplexed measurement of up to 800 genes in onereaction(24),using two 50-bp probes,which are hybridized to the targetmRNA in solution.This system does not rely on enzymatic amplification toproduce cDNA and is highly sensitive and reproducible,as demonstrated inprevious studies(25).To further demonstrate reproducibility,we analyzed theFO-mediated changes in MCP-1 and CD68 using both nanostring and con-ventional real-time RT-PCR.Similar changes were observed with both tech-niques,and the effect size was no different between these different methods.Comparing nanostring to real-time RT-PCR,the effect size for MCP-1 was0.539 and 0.524,and for CD68,the effect size was 0.595 and 0.518,respectively.For these studies,we designed a custom“chip”containing 116 genes of in-terest(Supplementary Table 1)in adipose tissue,and data were expressed inrelation to six different constitutive probes.Measurement of adipose tissue and muscle fatty acids.Lipids wereextracted from tissues(adipose,100 mg;muscle,20 mg)using acidified organicsolvents with the addition of 50 pmol of analysis-specific internal standards,including C15 fatty acid,synthetic glycerophospholipids incorporating a C17fatty acid,and mass-labeled di-and triglycerides.After evaporation to dryness,the material was dissolved in 1 mL 4:1 MeOH:CHCl3,and an aliquot was re-moved for phosphorous determination after wet digestion in perchloric acid(26).A portion of this material was saponified using methanolic KOH,and thereleased fatty acids were recovered and converted to their 3-acyloxymethyl-1-methylpyridinium iodide(AMMP)derivatives(27)using methods describedpreviously(28).In brief,the extracted saponified material was evaporated todryness and derivatized by reaction with 2-bromo-1-methylpyridinium iodideand 3-carbinol-1-methylpyridinium iodide.AMMP derivatives were analyzed byhigh-performance liquid chromatography electrospray ionization tandem massspectrometry using an ABSciex 4000 Q-Trap hybrid linear ion trap triplequadrupole mass spectrometer operated in positive mode with ion source.Instrument settings were optimized using a set of AMMP-derivatized fatty acidstandards.Fatty acid molecular species were quantitated by selected ionmonitoring using fatty acid speciesspecific precursor product ion transitions.Calibration was accomplished by reference to a set of synthetic fatty acidAMMP derivatives prepared from accurate mass standards.Where necessary,product ion spectra were generated for the most abundant species to de-termine the position of double bonds.For adipose tissue,data were expressedper tissue weight;although essentially identical results were obtained ifexpressed relative to oleate(the most abundant lipid).For muscle,the amountof available tissue was low,and tissue weight was less precise;therefore,datawere normalized to oleate.Tissue culture.THP-1 cells were polarized to differentiate into M1,M2a,andM2c macrophages,as described previously(5).In brief,THP-1 monocytes weredifferentiated into M1 macrophages using 100 ng/mL lipopolysaccaride(R&DSystems,Minneapolis,MN)and 20 ng/mL INF-g(R&D Systems),and into M2macrophages using 25 nmol/L phorbol ester(12-O-tetradecanoylphorbol-13-acetate)(Sigma-Aldrich,St.Louis,MO),followed by 20 ng/mL of either IL-4(R&D Systems)for M2a
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