Inflammatory Response in White Adipose Tissue in the Non-Obese

Hormone-Sensitive Lipase Null Mouse Model

Ola Hansson,*,† Kristoffer Stro1m,† Nuray Gu1 ner,‡ Nils Wierup,† Frank Sundler,†

Peter Ho1glund,‡ and Cecilia Holm†

Department of Experimental Medical Science, Lund University, BMC C11, SE-221 84 Lund, Sweden, andDepartment of Clinical Sciences, Lund University Hospital, Lund, Sweden

Received March 17, 2006

In the present study, a local inflammatory response in white adipose tissue from the nonobese HSL-null mouse model is demonstrated. The protein levels of several well-known markers of inflammation,like TNFR and ferritin HC, were highly increased and accompanied by an activation of NFκB. A numberof macrophage proteins, i.e., gal-3, Capg, and MCP-4, were expressed at increased levels andimmunohistochemical analyses revealed an increased infiltration of F4/80+ cells.

Keywords: hormone-sensitive lipase • white adipose tissue • proteomics • retinoic acid • inflammation

Introduction

Hormone-sensitive lipase is a key enzyme in the mobilizationof fatty acids from lipid stores in adipocytes as well asnonadipocytes. Expression of HSL has been demonstrated invarious tissues, including white adipose tissue (WAT),1 brownadipose tissue (BAT),2 myocytes,1 pancreatic â-cells,3 and testis,4

with the highest level in WAT. One distinctive feature of HSLamong lipases is its broad substrate specificity. Besides acyl-glycerols,5 HSL has the ability to hydrolyze cholesteryl esters,5

steroid esters,6 and retinyl esters.7 Several HSL-null mousemodels have been created in recent years.8-11 Among thedescribed characteristics of these models are male sterility,8-10

diglyceride accumulation in various tissues,9 and resistance todiet-induced obesity.12,13 These features have all been verifiedin the HSL-null mouse model investigated here, which hasfurthermore been shown to exhibit moderate impairment ofinsulin sensitivity in all insulin target tissues investigated, i.e.,liver, skeletal muscle, and WAT11 and unpublished results. Thisimpairment was manifested as perturbation of the ability ofinsulin to suppress hepatic glucose production, reduced insulin-stimulated glucose uptake in skeletal muscle and reduction ofinsulin-stimulated lipogenesis in WAT.11

The link between obesity and insulin resistance is wellestablished and growing evidence also connects inflammationto obesity, insulin resistance, and type 2 diabetes mellitus.14

In WAT of obese individuals increased expression of a numberof proinflammatory molecules, including interleukin-6,15 trans-forming growth factor-beta,16 and tumor necrosis factor-alpha(TNF-R),17 has been reported. However, the source of thesemolecules has been debated. It has been suggested that theyare secreted by adipocytes, by infiltrating inflammatory cells,

e.g., macrophages, or by both. A strong association betweenobesity and increased number of bone marrow-derived mac-rophages has been reported.18 In this study, the authors esti-mated that the percentage of macrophages in adipose tissueranges from 10% in lean mice to over 50% in extremely obese,leptin-deficient, mice.18 This estimation was based on a globalanalysis of the transcriptome, showing that a large number ofthe significantly regulated genes were macrophage and inflam-matory genes. However, one critical question is how the inflam-matory response is triggered and maintained in WAT in insulinresistant and obese states and what mechanisms could attractmacrophages to WAT. It has previously been reported that themRNA levels of TNFR are increased in WAT from HSL-nullmice, suggesting that there is WAT inflammation in this non-obese, insulin-resistant model.12 To characterize the inflam-matory state of this mouse model further, we chose to inves-tigate the protein expression profile of WAT using two-dimen-sional polyacrylamide gel electrophoresis (2D-PAGE). From amethodological point of view, analyzing data generated by 2D-PAGE involves many steps, including spot detection, spotmatching, background subtraction, and normalization. In theprocess of data analysis, a decision has to be made concerningthe combination of methods that are the most suitable to applyto the data set in order to get reliable quantification of proteins.This is an important issue that is often ignored and inad-equately documented in 2D-PAGE publications. When analyz-ing 2D-PAGE data, it is also important to consider how to han-dle missing data points. Is it a biological or a technical reasonfor the missing data points? Both these issues have beeninvestigated and are reported in the supplementary section ofthis paper.

The aim of this study was to investigate the protein expres-sion profile of WAT from HSL-null mice in comparison withwild-type littermates, fed either normal chow diet (ND) or highfat diet (HFD), to further explore the role of HSL in WAT biologyand inflammation.

* To whom correspondence should be addressed. Department of Experi-mental Medical Science, Division of Diabetes, Metabolism and Endocrinol-ogy, Lund University, BMC C11, SE 221 84 Lund, Sweden. Tel: +46 462229772. Fax: +46 46 2224022. E-mail: Ola.Hansson@med.lu.se.

† Department of Experimental Medical Science, Lund University.‡ Department of Clinical Sciences, Lund University Hospital.

10.1021/pr060101h CCC: $33.50 2006 American Chemical Society Journal of Proteome Research 2006, 5, 1701-1710 1701Published on Web 05/27/2006

Experimental SectionAnimals. The animals used in this study had a mixed genetic

background from the inbred strains C57BL/6 J and SV129.11

The animals were fed either ND or HFD prior to the time ofsacrifice. In the ND, 10.6% of the energy originated from fatand 73.1% from carbohydrate. In the HFD, 58.0% of the energyoriginated from fat and 25.6% from carbohydrate (Researchdiets incorporated). The mice had free access to food andwater at all times. When sacrificed the animals were anaes-thetized using midazolam 0.4 mg/mouse (Dormicum, Hoff-man-La-Roche, Basel, Switzerland) in combination with flu-anison 0.9 mg/mouse and fentanyl 0.02 mg/mouse (Hypnorm,Janssen, Beerse, Belgium) and killed with cervical dislocation.The studies were approved by the local Animal Ethics Com-mittee.

One-Dimensional Gel Electrophoresis. Parametrial WATfrom 13-months old female mice, fed a HFD for 40 weeks priorto the time of sacrifice, was homogenized using a Potter-Elvehjem homogenizer (400 rpm, 10 strokes). The samples werecentrifuged (10 000 × g, 4 °C, 30 min) and 100 µg of total proteinfrom the pellet fractions were separated using one-dimensionalpolyacrylamide gel electrophoresis (1D-PAGE) on a gradientgel (10-20%). The gel was stained according to.19 Briefly, thegel was fixed for 1 h in acetic acid, methanol and H2O (70:500:430) and then stained for 3 h in 2% phosphoric acid, 10%ammonium sulfate, 0.1% coomassie brilliant blue G-250:MeOH(4:1). After washing in deionized water, bands were excised andidentified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Two-Dimensional Gel Electrophoresis. Epididymal fat padsfrom four 12-14 months old male mice, fed either a ND or aHFD for 31 weeks prior to the time of sacrifice, from each groupwere dissected, weighed and snap frozen in liquid nitrogen.Homogenization was performed using a Potter-Elvehjemhomogenizer (400 rpm, 10 strokes) in a sample solutioncontaining 7 M urea, 2 M thiourea, 1% (w/v) dithiothreitol, 2%(w/v) CHAPS and 1.0% (v/v) IPG buffer 3-10. The samples werethen sonicated for 2 × 4 s, shaken for 1 h at 30 °C andcentrifuged (30 000 × g, 30 °C, 30 min). The clear infranatantwas recovered and total protein was measured with 2-D Quantkit (Amersham biosciences). Samples were stored in aliquotsat -80 °C until analysis. Samples were thawed for 30 min (30°C, 1000 rpm) before isoelectric focusing (IEF). ImmobilineDryStrips (11 and 17 cm, pH 3-10 NL, Bio-Rad) were used forIEF. Prior to IEF, each strip was rehydrated in 200 or 300 µL ofrehydration solution containing 100 µg protein for analyticalgels, 200-500 µg protein for preparative gels or 50 µg forWestern blot analysis. The rehydration solution consisted of 8M urea, 15 mM dithiothreitol, 0.5% (w/v) CHAPS and 1% (v/v)IPG buffer 3-10. Strips were allowed to rehydrate overnightunder a layer of mineral oil at 20 °C and 50 V in a Protean IEFCell (BioRad). Focusing was carried out at 200 V for 1 h, 500 Vfor 1 h, 1000 V for 1 h, 1000-8000 V over 30 min, and then8000 V for 35 kVh, or 25 kVh for 11 cm strips used in the westernblot analysis, to reach steady state. Following IEF the strips wereequilibrated for 15 min in a solution containing 6 M urea, 30%(w/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl pH 8.8 and 65mM dithiothreitol. In a second step, the strips were equilibratedfor an additional 15 min in the same solution except thatdithiothreitol was replaced by 260 mM iodoacetamide. Allsecond dimension runs were performed either in an EttanDaltsix electrophoresis system (Amersham biosciences) or inan Ettan Dalttwelve electrophoresis system (Amersham bio-

sciences) according to the manufacturer’s recommendations(12.5%T, 0.8% C, continuous). All strips were sealed at the topof the second dimension gels with 0.5% agarose and runovernight until the tracking dye had reached the anodic end.

Staining, Image Acquisition, and Statistics. Gels werestained with SYPRO Ruby protein gel stain (Bio-Rad) accordingto the manufacturer’s recommendations. Briefly, gels were fixedin 40% ethanol and 10% acetic acid for 3 h, stained overnightin SYPRO Ruby protein gel stain, washed in 10% methanol and7% acetic acid for 30-60 min and scanned with a FLA 3000gel scanner (Fuji film) at 50 µm resolution (532 nm, O580,F1000). The 2D-PAGE image computer analysis was carried outusing the Phoretix 2D v2002.01 software package (Nonlineardynamics). Spots were detected and matched automatically andthen manually controlled. Background subtraction was per-formed using the method of average on boundary and relativevolumes were calculated with normalization to total volumeof spots present in the individual gel to correct for staining andloading differences. Only spots that were present in all gels wereused for normalization. Duplicate samples were analyzed. Ifboth samples produced quantifiable spots, then the mean valuewas used, and if only one of the two spots could be measured,then that observation was used in the calculations. If none waspresent, the data point was regarded as missing and set to zero.Statistical analyses were performed using the nonparametricSpearman’s Rho, McNemar’s, Kruskal-Wallis and Mann-Whitney U tests. Further details of the 2D-PAGE data analysisprocess are given in the Supporting Information.

Identification and Characterization of Proteins in Spots.Preparative 2D-PAGE, spot excision, protein digestion and massspectrometry were performed at the Swegene proteomicsresource center in Lund (http://www.swegene.org/proteomics).Briefly, protein spots that appeared to be regulated based onthe image analysis were excised either using an Ettan Spothandling workstation (Amersham Biosciences) or using pipettips and transferred to original Eppendorf tubes. All followingsteps were handled using an Ettan Spot handling workstation(Amersham Biosciences). The gel pieces were destained with50% acetonitrile, 25 mM NH4HCO3, and dried using a speedvac concentrator for 10-15 min. Digestion was performedovernight at 37 °C with 12.5 µg/mL trypsin (Promega) in 50mM NH4HCO3. The trypsin solution was added in a volumesufficient to cover the gel piece (∼10 µL). The digestion wasterminated and the peptides extracted by addition of 5%trifluoroacetic acid in 75% acetonitrile in an equal volume asthe trypsin solution. Sample (0.5 µL) was added to the tar-get plate (Waters) and allowed to dry, then equal volume(0.5 µL) of matrix solution (5 mg/mL R-cyano-4-hydroxycin-namic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroaceticacid) and sample (0.5 µL) was eluted onto the plate and allowedto dry.

Mass Spectrometry. Mass spectrometry was performed usinga MALDI-TOF mass spectrometer (MALDI LR HT, Waters). Eachspectrum represented up to 200 laser shots, depending on thesignal-to-noise ratio. The resulting mass spectra were internallycalibrated using the auto-digested tryptic mass values visiblein all spectra. Calibrated spectra were processed and peaksextracted by the Piums software.20 Multiple searches were doneusing the automated Piums software.21 Database searching wasalso performed using the MASCOT 2.0.0 software. Sinceproteins were recovered from gels, carbamidomethylation wasset as a fixed modification and methionine oxidation as avariable one. The peptide tolerance was set to 50-200 ppm in

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the Piums software and to 200 ppm in the MASCOT software.Only one trypsin missed cleavage was allowed. The proteinidentifications were considered to be confident when theexpectancy score was below 0.001. Data from the identificationprocess is presented in table S3 as supplement information.The average sequence coverage of the identified proteins wasapproximately 43%. The molecular mass and pI of the identifiedproteins were evaluated by analysis of the mobility of thecorresponding protein spot in the 2D-PAGE images.

Western Blot Analysis. Parametrial WAT from 13 to 15months old female mice, fed either a ND or a HFD for 36-40weeks prior to the time of sacrifice, were dissected and snapfrozen in liquid nitrogen and then homogenized using aPotter-Elvehjem homogenizer (400 rpm, 10 strokes) in 0.25 MSucrose, 1 mM EDTA, pH 7.0, 1 mM dithiothreitol, 20 µg/mLleupeptin, 10 µg/mL antipain and 1 µg/mL pepstatin A. Thesamples were centrifuged at 10 000 × g for 25 min at 4 °C.Aliquots of the infranatant and pellet fractions were collectedand stored at -20 °C until analyzed further. Protein concentra-tion was determined using BCA-assay (Pierce). Proteins wereresolved by either 1D-PAGE or 2D-PAGE and electroblotted tonitrocellulose membranes. Detection of protein was accom-plished using: a monoclonal rat anti human galectin-3 anti-body (a kind gift from Dr. Hakon Leffler, Lund University,Sweden), a polyclonal goat anti mouse TNFR antibody (SantaCruz Biotechnology), a polyclonal rabbit anti human phospho-IκB-R (ser32) antibody (Cell Signaling Technology), a mono-clonal mouse anti rabbit glyceraldehyde-3-phosphate dehy-drogenase antibody (Chemicon international), a polyclonalrabbit anti mouse mast cell protease 4 antibody (a kind giftfrom Dr. Lars Hellman, Uppsala University, Sweden) and amonoclonal mouse anti rat pyruvate carboxylase antibody (akind gift from Dr. John Wallace, University of Adelaide,Australia). Monoclonal mouse anti chicken R-Tubulin antibody(Sigma) was used as loading control. Western blots weredeveloped using a CCD camera (LAS 1000, Fuji Film).

Real Time Quantitative PCR. Parametrial WAT from 13 to15 months old female mice fed HFD for 36-40 weeks prior tothe time of sacrifice was dissected and snap frozen in liquidnitrogen. For quantification of F4/80 mRNA 7 months oldfemale mice fed ND or HFD for 12 weeks prior to the time ofsacrifice were used. Total RNA was isolated and purified usingRNeasy Lipid Tissue Mini Kit (Qiagen) according to themanufacturer’s recommendations. RNA integrity was verifiedwith agarose gel electrophoresis. Total RNA (1 µg) was treatedwith DNase I (DNase I amplification grade, Invitrogen) andthen reversely transcribed using random hexamers (AmershamBiosciences) and SuperScriptTMII RNaseH reverse transcriptase(Invitrogen Life Technologies) according to the manufacturer’srecommendations. The mRNA levels of spot14 (Assays-on-demand, Mm00493680_s1), glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) (Assays-on-demand, Mm99999915_g1)and F4/80 (Assays-on-demand, Mm00802530_m1) were quan-tified using TaqMan real-time PCR with an ABI 7900 system(Applied Biosystems). As a control to normalize gene expres-sion, ribosomal protein 29s was used in SYBR green chemistry(forward primer: 5′-GGA GTC ACC CAC GGA AGT-3′ andreverse primer: 5′-TCC ATT CAA GGT CGC TTA GTC-3′).Omitting reverse transcriptase in the reactions checked theabsence of contamination by genomic DNA. Each sample wasanalyzed in duplicates.

Immunohistochemistry. Parametrial WAT from 9 to 10months old female mice, fed either a ND or a HFD for 24 weeks

prior to the time of sacrifice, was fixed overnight in 4% bufferedparaformaldehyde, pH 7.2, dehydrated in graded ethanols, andembedded in paraffin. Sections (6 µm) were mounted on slidesand deparaffinized. The sections were incubated overnight at4 °C with a monoclonal rat anti mouse F4/80 antibody(Serotec), diluted 1 to 200 in PBS (pH 7.2) containing 0.25%BSA and 0.25% Triton-X 100. After a rinsing step, sections wereincubated with anti rat IgG coupled to Texas-Red (Jackson,West Grove, PA, US) for 1 h at room temperature. After a secondrinsing step, sections were mounted in PBS/glycerol (1/1).Immunofluorescence was examined in an epifluorescencemicroscope (Olympus BX60) and images were captured with adigital camera (Olympus DP50).

Cellular Fractionation. Periovarial WAT from 7 months oldfemale mice, fed a ND or HFD for 12 weeks prior to the timeof sacrifice, was excised, cut into small pieces and incubatedin Krebs-Ringer solution (pH 7.4) supplemented with 3.5%BSA, 2 mM glucose, 200 nM adenosine and collagenase (1mg/mL; Sigma) in a shaking incubator at 37 °C for ∼60 min,according to a modification22 of the Rodbell method.23 Thedigested tissue was filtered and the isolated cells were washedtwice in Krebs-Ringer buffer (pH 7.4) with 1% BSA, 200 nMadenosine and 2 mM glucose by allowing the isolated adipo-cytes to float to the surface and then aspirate the underlyingbuffer. The aspirated buffer from the two washing steps waspooled and subjected to a brief centrifugation at 200 × g for 5min, and the supernatant was removed, yielding a pelletcontaining the stromal-vascular fraction (SVF). Total RNA wasisolated, purified, and analyzed as described above.

Subcellular Fractionation. Parametrial WAT from 8 monthsold female mice fed HFD for 24 weeks prior to the time ofsacrifice were dissected and homogenized using a Potter-Elvehjem homogenizer (400 rpm, 10 strokes) in 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 250 mM Sucrose, 20 µg/mLleupeptin, 10 µg/mL antipain and 1 µg/mL pepstatin A. Toremove cell debris and the fat cake, a brief centrifugation (5min/80 g) was performed and the infranatant was removedusing a syringe. The infranatant was then centrifuged at 720 gfor 10 min and the resulting supernatant was discarded. Thepellet was washed in a small volume of TES buffer and thencentrifuged at 720 g for an additional 5 min. After removal ofthe supernatant the pellet, containing the nuclear fraction, waslysed in TES buffer supplemented with 5% SDS in a shakingincubator (Eppendorf) at 50 °C for 30 min and the proteinconcentration was determined using BCA-assay (Pierce). A 5-µgportion of protein from the nuclear fraction were separatedusing 1D-PAGE and the amount of GAPDH protein wasmeasured using western blot analysis as described above fromthree individuals of each genotype.

Retinyl Ester Hydrolase Activity. Parametrial WAT from 13to 15 months old female mice fed ND or HFD for 36-40 weeksprior to the time of sacrifice were dissected and tissues werehomogenized using a Potter-Elvehjem homogenizer (400 rpm,10 strokes) in 0.25 M sucrose, 1 mM EDTA, pH 7.0, 1 mMdithiothreitol, 20 µg/mL leupeptin, 10 µg/mL antipain, and 1µg/mL pepstatin A, followed by a centrifugation at 10 000 × gfor 25 min at 4 °C. The fat cake was discarded and theinfranatant was separated from the pellet. Retinyl ester hydro-lase activity was measured using retinyl palmitate (RP) assubstrate. The method is based on measurements of releaseof [14C] palmitate from retinyl-[14C]palmitate (American Radio-labeled Chemicals Inc.) and was adapted from previouslydescribed methods utilizing phospholipid-stabilized emulsions

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of triolein, diolein and cholesterol oleate.24 Briefly, [14C]RP (1µCi) and unlabeled RP (together corresponding to 990 µg) wasemulsified with 0.6 mg of phospholipids (phosphatidylcholine/phosphatidylinositol, 3:1, w/w) in 20 mM KH2PO4 (pH 7.0), 1mM EDTA, 1 mM dithioerythritol, 0.02% defatted BSA usingsonication, to yield a final substrate concentration of 0.5 mM.This concentration has previously been shown to yield Vmaxconditions,7 which was confirmed in our assay system. One unitof enzyme activity is equivalent to 1 µmol of fatty acidsreleased/min at 37 °C. Specific activity is expressed as mU/mgof protein.

Results

1D-PAGE Protein Expression Profile of WAT from HSL-NullMice Fed HFD. To compare the protein expression in WATfrom HSL-null mice to that of wildtype littermates, proteins ofcrude homogenates were separated using 1D-PAGE. Followingstaining with coomassie brilliant blue, five differentially ex-pressed proteins were identified using MALDI-TOF-MS. Twoof these were found to have decreased expression levels, i.e.,pyruvate carboxylase (PC) and polymerase I, and three in-creased expression levels, i.e., 94 kDa glucose-regulated protein(grp94), 78 kDa glucose-regulated protein and mast cell pro-tease 4 (MCP-4), in HSL-null mice compared to wild-typelittermates. The expression change for PC (0.15, p < 0.01) andMCP-4 (1.7, p < 0.05, Figure 1) was verified using 1D Westernblot analysis.

2D-PAGE Protein Expression Profile of WAT from HSL-NullMice Fed either ND or HFD. Having successfully identified fiveproteins with differential expression using 1D-PAGE separation,a more comprehensive protein expression profiling was per-formed using 2D-PAGE. In this analysis, duplicate gels fromfour individuals in each group were generated. A reference gelwas created with 1026 matched spots. A representative geltogether with enlargements of spots found to be differentiallyexpressed is displayed in Figure 2. A series of Kruskal-Wallistests were applied in order to identify the spots that hadsignificant changes in their expression levels. In total, 297 spotswere found to have a significantly changed expression level (p< 0.05). This is far more than the 50 spots, which are expectedby chance alone on a 0.05-level. To be able to determinebetween which groups the differences were manifested, pair-wise comparisons using Mann-Whitney U tests were alsoperformed (p < 0.05). The number of differences found in thecomparisons made was as follows: wild-type ND vs HSL-nullND (85 spots), wild-type HFD vs HSL-null HFD (140 spots),wild-type ND vs wild-type HFD (61 spots) and HSL-null ND vsHSL-null HFD (52 spots). The results indicate a larger influence

of the genotype than of the diet (85 and 140 vs 61 and 52 spots)on regulation of protein expression. Furthermore, they showthat the expression differences between the two genotypesexamined were more pronounced in the HFD groups than inthe ND groups (140 spots vs 85 spots). Following identificationof proteins with MALDI-TOF-MS, they were categorized ac-cording to their function (Table 1). In the category of metabo-lism two members of the acyl-CoA dehydrogenase proteinfamily were found to be down-regulated in HSL-null micecompared with wild-type littermates in both diet groupsstudied, i.e., isovaleryl-CoA dehydrogenase (0.39 and 0.48, p <0.05) and short chain acyl-CoA dehydrogenase (SCAD) (0.59and 0.68, p < 0.05). SCAD was also found to be down-regulatedin wild-type mice fed HFD in comparison with wild-type micefed ND (0.84, p < 0.05). In connection to this finding, adecreased expression level of electron transferring flavoproteinalpha (ETFR) was observed in HSL-null mice compared withwild-type littermates in both diet groups studied (0.53 and 0.49,p < 0.05). Spot14, another protein involved in fatty acidmetabolism, was detected in all groups examined with theexception of HSL-null mice fed HFD. The decrease in expres-sion level of spot 14 in the HSL-null HFD group was verifiedusing real time quantitative PCR (rtPCR) (0.31 HSL-null HFDvs wild-type HFD, p < 0.05). One exception to the observeddown-regulation of proteins involved in fatty acid metabolismin HSL-null mice was found, i.e., ∆3,5,∆2,4-dienoyl-CoA isomerase(DI) (1.25 HSL-null ND vs wild-type ND, p < 0.05). DI is anauxiliary enzyme of unsaturated fatty acid â-oxidation. Thelargest difference found in the 2D-PAGE analysis was an up-regulation of GAPDH. Two spots were identified as GAPDH,both being upregulated in HSL-null mice compared with wild-type littermates, regardless of diet (13.13, 6.92 and ON, ON, p< 0.05) (Table 1). Attempts were made to verify the change inexpression with rtPCR and 1D Western blot analysis, but nosignificant increase in the expression of GAPDH was detected(data not shown), on the contrary a significant decrease wasfound at the mRNA level (0.55 HSL-null HFD vs wild-type HFD,p < 0.05). In a 2D Western blot analysis, a signal correspondingto the up-regulated spots identified as GAPDH in the 2D-PAGEprotein expression profile were detected in samples corre-sponding to WAT from HSL-null mice from both diet groupsinvestigated, but it was not detected in samples correspondingto WAT from wild-type mice (Figure 3). To investigate thelocalization of GAPDH a subcellular fractionation was per-formed of WAT from HSL-null mice and wild-type littermatesfed HFD. Using 1D Western blot analysis, an approximately10-fold increase in the amount of GAPDH was detected in thenuclear fraction of WAT from HSL-null mice compared to wild-type littermates (data not shown). Four proteins were catego-rized as being involved in inflammation, i.e., macrophage-capping protein g (capg), annexin A2, galectin-3 (gal-3), andferritin heavy chain (ferritin H). Capg was found to have anincreased expression level in WAT from HSL-null mice fed NDto the same level as wild-type mice fed HFD in comparisonwith wild-type mice fed ND (2.95 and 3.04 respectively, p <0.05). Capg is an abundant protein in macrophages and isbelieved to be involved in actin function. Annexin A2 isexpressed on the surface of macrophages and has been shownto be a binding site for plasminogen.25 It has also recently beenreported that annexin A2 is a thiazolidinedione-responsive geneinvolved in glucose transporter 4 translocation in 3T3-L1adipocytes.26 A reduction in the expression of annexin A2 wasobserved in HSL-null mice in both diet groups, compared with

Figure 1. Protein level in WAT of mast cell protease 4 in 13-15months old female mice, fed HFD for 36-40 weeks measuredusing 1D Western blot. Values given are mean fold change overcontrol ( SEM, * indicates p < 0.05 analyzed with Mann-Whitney U test, n ) 4-5.

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wild-type littermates (0.31 and 0.35 respectively, p < 0.05).Gal-3 is an important mediator of inflammation and is ex-pressed in activated macrophages. The spot identified as gal-3was found in 2D-gels corresponding to WAT from HSL-nullmice from both diet groups examined, but it was not detectedin samples corresponding to WAT from wild-type mice. Atendency toward a higher expression level in HSL-null mice

fed HFD compared to HSL-null mice fed ND was detected, butthis difference was not statistically significant. The increasedexpression level of gal-3 in HSL-null mice was confirmed (4.37,p < 0.001, HSL-null ND vs wild-type ND and 1.93, p < 0.05HSL-null HFD vs wild-type HFD) using 1D Western blotanalysis (Figure 4). An inflammatory response in wild-type micefed HFD was also indicated by an increased protein level of

Figure 2. Representative SYPRO Ruby stained 2D-PAGE gel images of WAT. The total protein load was 100 µg. For separation in thefirst dimension a nonlinear pH 3-10 Immobiline DryStrip was used and for the second dimension a continuous 12.5% T, 0.8% Cpolyacrylamide gel was used. The gels display 1026 matched spots. Representative enlargements of spots found to be differentiallyexpressed are presented.

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gal-3 in wild-type mice fed HFD compared with wild-type micefed ND (2.69, p < 0.01). Another indication of an inflammatoryresponse in WAT from HSL-null mice was a large increase inthe expression level of ferritin H in WAT from HSL-null mice(8.44, p < 0.05, HSL-null ND vs wild-type ND and 2.74, p <0.05 HSL-null HFD vs wild-type HFD). Ferritin is the majoriron-binding protein in eukaryotic cells. It is composed of 24subunits of ferritin H and ferritin light chain (ferritin L),assembled in various ratios in different tissues and diseasestates, including inflammation.27 Elevated serum level of ferritinis a characteristic of states of chronic inflammation and it haspreviously been shown that the expression of ferritin H can be

induced by TNFR.28 Furthermore, an increase in the expressionlevel of ferritin H was also detected when comparing WAT fromwild-type mice fed HFD and wild-type mice fed ND (3.17, p <0.05). A down-regulation of ferritin L was detected in WAT fromHSL-null mice fed either ND or HFD compared with wild-typelittermates (0.49, p < 0.05, HSL-null ND vs wild-type ND and0.37, p < 0.05 HSL-null HFD vs wild-type HFD). No significantdifference in the expression of ferritin L was detected in WATfrom wild-type mice fed HFD compared with wild-type micefed ND. Aldehyde dehydrogenase is an enzyme involved in theconversion of retinol to retinoic acid. The expression level ofthe enzyme was found to be increased in HSL-null mice fedHFD in comparison with wild-type mice fed HFD (1.41, p <0.05), whereas a decreased level was observed when comparing

Table 1. Differentially Expressed Proteins in WAT from HSL-Null Mice, Fed Either ND or HFD, Compared with Wild-TypeLittermatesa

ratio

protein

Swiss Prot

ac. no.

HSL-null ND vs

wild-type ND

HSL-null HFD vs

wild-type HFD

wild-type HFD vs

wild-type ND

HSL-null HFD vs

HSL-null ND

Metabolismisovaleryl-CoA dehydrogenase Q9JHI5 0.39 N. C. 0.48 N. C.SCAD Q07417 0.59 0.68 0.84 N. C.∆3,5,∆2,4-dienoyl-CoA isomerase O35459 1.25 N. C. N. C. 0.52spot14 Q62264 N. C. OFF N. C. OFFaldehyde dehydrogenase P47738 N. C. 1.41 0.63 N. C.ETF alpha Q99LC5 0.53 0.49 N. C. N. C.GAPDH P16858 ON ON s N. C.GAPDH P16858 13.13 6.92 N. C. N. C.carbonyl reductase 3 Q8K354 0.29 N. C. N. C. N. C.carbonic anhydrase III P16015 0.38 N. C. 0.57 N. C.carbonic anhydrase III P16015 0.28 N. C. 0.32 N. C.heme binding protein 1 Q9R257 0.28 N. C. N. C. N. C.ferritin light chain 1 P29391 0.49 0.37 N. C. N. C.

Inflammationcapg protein Q99LB4 2.95 N. C. 3.04 N. C.annexin A2 P07356 0.31 0.35 N. C. N. C.galectin-3 P16110 ON ON s N. C.ferritin heavy chain P09528 8.44 2.74 3.17 N. C.

Otherschloride intracell. channel pr. 1 Q9Z1Q5 4.86 2.18 2.76 N. C.dual specificity pp 3 Q9D7 × 3 N. C. 0.40 N. C. N. C.inorganic pyrophosphatase Q9D819 0.36 N. C. 0.50 N. C.HSP60 P63038 0.53 0.52 N. C. N. C.actin, cytoplasmic 2 P63260 3.78 3.33 N. C. N. C.apolipoprotein A-I Q00623 2.66 N. C. N. C. N. C.vimentin P20152 10.78 OFF N. C. OFFvimentin P20152 0.36 N. C. N. C. 4.18

a p < 0.05, Mann-Whitney U tests. No change (N. C.), not expressed, but expressed in the control group (OFF), expressed, but not in the control group(ON), not expressed in either group (-).

Figure 3. Representative 2D Western blots of glyceraldehyde-3-phosphate dehydrogenase of WAT from wildtype mice fed ND(A) or HFD (B) and from HSL-null mice fed ND (C) or HFD (D).Arrows indicate the signals corresponding to the differentiallyexpressed spots identified as GAPDH in the 2D-PAGE proteinexpression profile. n ) 2-3.

Figure 4. Protein level in WAT of galectin-3 (gal-3) in 13-15months old female mice, fed either ND or HFD for 36-40 weeksmeasured using 1D Western blot. R-tubulin was used as aninternal control, n ) 6-7. Values given are mean ( SEM, *indicates p < 0.05, ** indicates p < 0.01 and *** indicates p <0.001 analyzed with Mann-Whitney U test.

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wild-type mice fed HFD with wild-type mice fed ND (0.63,p < 0.05).

Increased Level of TNFR in WAT from HSL-Null Mice FedHFD. As the protein expression profile indicated an increasedexpression of inflammation-related proteins, i.e., ferritin H andgal-3 among others, the level of TNFR was investigated in WATfrom HSL-null mice and wild-type littermates fed HFD using1D Western blot analysis. TNFR is an important cytokine andmediator of inflammation and the protein level gives anindication of the inflammatory status of the animals. TNFRconsists of a membrane bound 26 kDa protein, which can becleaved to a soluble and secreted 17 kDa form.29 In WAT fromHSL-null mice fed HFD a severalfold increase in the levels ofboth isoforms was observed, 4.03 p < 0.01 and 3.51 p < 0.01,respectively (Figure 5). This increase of TNFR expression is inagreement with previous findings where an increase of TNFRmRNA level has been reported in HSL-null mice fed either aND or a HFD.12

Increased Activation of NFκB in WAT from HSL-Null MiceFed HFD. Nuclear factor kappa B (NFκB) is a nuclear transcrip-tion factor regulating the expression of many proinflammatorygenes, including TNFR. Activation of NFκB was measuredindirectly by measuring the phosphorylation level of IκB-R, aninhibitor of NFκB that is inactivated upon phosphorylation ofSer-32. Using 1D Western blot analysis and a phosphospecificantibody, a severalfold increase in phosphorylation at Ser-32of IκB-R was observed (3.47, p < 0.01) in WAT from HSL-nullmice fed HFD compared with wild-type littermates (Figure 6).

Macrophage Infiltration of WAT from HSL-Null Mice Fedeither ND or HFD. To assess the degree of macrophage

infiltration in WAT, immunohistochemistry was performedusing antibodies against F4/80, a commonly used and specificmacrophage marker. An increased F4/80 signal in sections ofWAT from HSL-null mice was detected compared to wild-typelittermates (Figure 7). This sign of increased infiltration ofmacrophages was found in both the ND and the HFD group,indicating increased inflammation in WAT of HSL-null miceregardless of diet. The mRNA level of F4/80 was also investi-gated using rtPCR. To investigate the location of the F4/80positive cells, WAT was fractionated in an adipocyte fractionand a SVF prior to the rtPCR analysis. The SVF contained alarger amount of F4/80 mRNA then the adipocyte fraction,irrespective of either genotype or diet (data not shown). Asignificant increase in SVF of F4/80 mRNA was observed whencomparing wild-type mice fed HFD with wild-type mice fedND (2.10, p < 0.05) (Figure 8). Furthermore, the SVF from HSL-null mice contained larger amounts of F4/80 mRNA comparedwith the corresponding fraction from wild-type littermates inboth the ND (4.29, p < 0.05) and the HFD (4.70, p < 0.05) group.

Figure 5. Protein level in WAT of TNFR in 13-15 months oldfemale mice, fed HFD for 36-40 weeks measured using 1DWestern blot. TNFR of 17 kDa corresponds to the released solubleform and TNFR of 26 kDa corresponds to the membrane-boundform. n ) 4-5, values given are mean fold change over control( SEM, * indicates p < 0.05 analyzed with Mann-Whitney U test.

Figure 6. Phosphorylation level of IκB in WAT from 13 to 15months old female mice, fed HFD for 36-40 weeks, analyzedusing 1D Western blot and phosphospecific antibodies (Ser 32).R-tubulin was used as an internal control, n ) 4-5. Values givenare mean fold change over control ( SEM, * indicates p < 0.05analyzed with Mann-Whitney U test.

Figure 7. Immunohistochemistry images of epididymal WATfrom 9 to 10 months old wild-type mice fed ND (A) or HFD (B)and from HSL-null mice fed ND (C) or HFD (D) for 24 weeks.Increased staining for the specific macrophage marker F4/80 (red)indicates a larger infiltration of this cell type in WAT from HSL-null mice in both diet groups investigated compared with wild-type littermates.

Figure 8. mRNA level of the macrophage marker F4/80 in thestromal-vascular fraction of WAT from 7 months old female mice,fed ND or HFD for 12 weeks, analyzed with rtPCR. n ) 4-5. Valuesgiven are mean ( SEM, * indicates p < 0.05 analyzed withMann-Whitney U test.

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This was also observed in the adipocyte fraction (data notshown).

Decreased Retinyl Ester Hydrolase Activity in WAT fromHSL-Null Mice Fed either ND or HFD. To test the hypothesisthat the inflammatory response in WAT of HSL-null mice isdue to a reduced ability to generate retinoic acid, which hasantiinflammatory properties,30 from retinyl ester stores theretinyl ester hydrolase activity was investigated. A significantdecrease in total retinyl ester hydrolase activity was found inhomogenates of WAT from HSL null mice fed either ND (0.23,p < 0.01) or HFD (0.23, p < 0.01) compared to wild-typelittermates. A significant decrease was also observed whencomparing wild-type mice fed HFD with wild-type mice fedND (0.64, p < 0.05) (Figure 9).

Discussion

The HSL-null mouse model investigated here exhibits insulinresistance at several sites, including adipose tissue, liver, andskeletal muscle11 and is protected against diet-induced obesity(manuscript in preparation). It has previously been reportedthat the mRNA level of TNFR is increased in WAT from HSL-null mice,12 suggesting an inflammatory response in the tissue.To further characterize the inflammatory state of this mousemodel, we chose to investigate the protein expression profileof WAT using 1D- and 2D-PAGE.

In the protein expression profile, several proteins involvedin fatty acid metabolism, in particular â-oxidation, wereobserved to have decreased expression in the HSL-null mice.Among the proteins found to have reduced expression wereSCAD, ETFR, and Spot14. SCAD is involved in â-oxidation offatty acids and ETFR is a protein that accepts electrons fromthe â-oxidation pathway and delivers them to the mitochon-drial electron transport chain. Spot14 is a protein highlyexpressed in triglyceride synthesizing tissues like liver, WAT,and BAT and is believed to play a role in lipogenesis. On onehand, this finding indicates a decreased capacity of HSL-nullmice to perform â-oxidation in WAT. On the other hand, outof several acyl-CoA dehydrogenases involved in â-oxidationSCAD was the only one found in the protein expression profileand SCAD is only responsible for degradation of short chainfatty acids (C4 and C6). Further experiments are needed in orderto clarify this aspect of the phenotype.

The largest increase in expression found in the 2D-PAGEanalysis was an up-regulation of GAPDH. The molecular weightof the increased protein was, however, approximately 3-5 kDalower than the expected size of 36 kDa. When attempting to

verify this change in expression with 2D Western blot analysisboth the full-length GAPDH and a smaller isoform weredetected in WAT from HSL-null mice, but only the full-lengthisoform was detected in WAT from wild-type mice. The natureof the smaller GAPDH variant is not known but there are severalpossibilities. It could represent a splice variant, a post-trans-lationally modified variant or a degradation product of the full-length protein. Although protein degradation may be the mostobvious explanation, the fact that peptides from both theC-terminal and N-terminal end of the protein were representedin the MALDI-TOF-MS identification of the protein arguesagainst this. GAPDH is known as a glycolytic enzyme, but inrecent years, it has been demonstrated that GAPDH is involvedin a number of diverse functions including apoptosis.31,32 Manyof these new functions include a translocation of the proteinto the nuclear compartment. The data presented here indicatesthat a larger amount of GAPDH is localized in the nucleus inHSL null mice compared with wild-type littermates. However,at this point we do not know if this corresponds to the smallerisoform or not. It has previously been shown that a truncationor mutation of a highly charged 13 amino acid nuclear exportsignal (NES) peptide in GAPDH results in a nuclear localizationof the protein.33 A perturbation of this NES could be anexplanation for the data presented concerning GAPDH. Theobservations made concerning GAPDH in the 2D-PAGE analysishighlights an important issue when interpreting data obtainedwith this technique. The observed expression changes may bea result of posttranslational modifications of the protein thatchange its properties without affecting the total expressionlevel. It is possible that other isoforms of the same proteindisplay changed expression without being identified in theanalysis. Because of this problem care should be taken beforemaking any assumptions regarding the biological importanceof such changes before appropriate verification experimentshave been made.

The present study demonstrates the presence of an inflam-matory response in WAT from the nonobese HSL-null mousemodel in both diet groups investigated. There are severalmanifestations of this inflammatory response, including in-creased protein levels of several well-known markers of inflam-mation, like TNFR and ferritin H, activation of NFκB, increasedexpression of several macrophage and mast cell proteins,including gal-3, capg, and MCP-4, and macrophage infiltrationin WAT. To determine at what age the inflammatory responsefirst is manifested more experiments are clearly needed.However, as the animals used in the fractionation experimentwere 7 months old the inflammatory response is present at leastfrom this age.

Many factors have been implicated to provide the linkbetween obesity and inflammation in WAT. Insulin is knownto have antiinflammatory effects and in insulin-resistant statesthese effects are perturbed. As the HSL-null mouse modelinvestigated here displays signs of insulin resistance at the levelof WAT and elevated plasma insulin levels,11 it is possible thatthis factor contributes to the described inflammatory response.Other factors to take into consideration are adipokines, suchas adiponectin (Acrp30). Acrp30 is a 30-kDa protein secretedfrom adipose tissue and decreased plasma level of this adi-pokine is normally observed in states of obesity. In mousemodels, where the level of Acrp30 is manipulated, a loweringof the expression level of Acrp30 is usually accompanied byan increased level of TNFR. One example is the Acrp30-nullmouse.34 Decreased WAT mRNA level of Acrp30 has previously

Figure 9. Total retinyl ester hydrolase activity in homogenatesof WAT from 13 to 15 months old female mice, fed either ND orHFD for 36-40 weeks. n ) 6-7. Values given are mean ( SEM,* indicates p < 0.05 and ** indicates p < 0.01 analyzed withMann-Whitney U test.

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been reported in HSL-null mice fed either ND or HFD12 andin the HSL-null mouse strain investigated here, decreasedplasma levels of Acrp30 protein have been observed in bothdiet groups investigated (manuscript in preparation). Onepossible explanation for the observed increase of TNFR in WATof HSL-null mice could be the decreased level of Acrp30.

The inflammatory response in WAT is presumably thecumulative result of many different events, rather than inducedby a single factor. A key question with regard to our model ishow the absence of the metabolic enzyme HSL triggers WATinflammation. At this point, we have no clear answer to thisquestion. However, one could speculate that a connectionbetween HSL expression and the regulation of target geneexpression could be that HSL provides ligands for transcriptionfactors such as PPARγ or any of the retinoid nuclear receptors.Keeping the broad substrate specificity of HSL in mind thisligand could be any of a number of candidates. An appealingcandidate is some derivate of vitamin A, i.e., an isomer ofretinoic acid (RA). Isomers of RA have previously been shownto exert anti-inflammatory and immunomodulatory effects.30

Furthermore, a reduced or absent vitamin A signal leads toactivation of NFκB, thereby possibly providing a link betweenvitamin A and an inflammatory response in WAT.35 The anti-inflammatory role of RA is further supported by findings of itsinhibitory effect on inflammatory cytokine production in anactivated macrophage cell line.36 Among the cytokines reportedto be inhibited by RA are nitric oxide, interleukin-1 beta andTNF-R.36 Gal-3 expression level has been shown to be sup-pressed by RA in a F9 cell line,37 thereby providing furthersupport for an anti-inflammatory role of RA. It has previouslybeen shown that adipocytes contain a significant amount ofthe total body store of retinyl esters. In fact, in rats, adiposetissue is the second largest depot after the liver and containsapproximately 15% of total retinyl esters present in the body.38

However, the mechanism behind the mobilization of thesestores has not yet been fully elucidated. It has been suggestedthat HSL may play a role in this mobilization of retinyl esterstores in WAT.7 The reduction of retinyl ester hydrolase activityin WAT from HSL-null mice (Figure 9), together with in-creased retinyl ester stores (manuscript in preparation), ob-served in our HSL-null mice clearly indicates that HSL couldhave this function in WAT. In 2002, Balmer and Blomhoffpublished a summary of 532 genes known to be regulated byRA.39 Among these are several of the proteins found to have adifferential expression in the protein expression profile pre-sented here, including spot14, apolipoprotein A1, gal-3, alde-hyde dehydrogenase, grp94, and vimentin, providing furthersupport for the hypothesis that HSL plays a role in RA-mediatedgene regulation.

Our working hypothesis is that in the absence of HSL inadipocytes, and possibly other cell types present in WAT, suchas preadipocytes, epithelial cells, or macrophages, a RA ligandis not generated to suppress NFκB activity. The increasedactivity of NFκB then leads to an increased level of TNFR inWAT. The possible decrease of RA in macrophages could alsogive rise to increased levels of other pro-inflammatory mol-ecules like nitric oxide and interleukin-1 beta. Furthermore,gal-3 is known to be involved in chemotaxis, i.e., attractinginflammatory cells to sites of inflammation and thereby con-tributing to the recruitment of macrophages and other inflam-matory cells to WAT. Many important questions still remainunanswered. One such is if RA has a role in the inflammatory

response in WAT in a normal setting, keeping in mind theextreme genotype that a null mouse model represents.

In conclusion, to analyze the data generated by 2D-PAGEwe have used measurements of the correlation between meansand standard deviation to evaluate different parts of the dataanalysis process and also presented a way to deal with theproblem of missing data by setting up a few simplistic rules.The present study has also demonstrated the presence of aninflammatory response in WAT from the nonobese HSL-nullmouse model, fed either a ND or a HFD.

During the preparation of this manuscript, Cinti and co-workers have demonstrated a 15-fold increase in necrotic-likeadipocyte death in an independently generated HSL-nullmouse strain fed normal chow diet. In agreement with the datapresented here, they also show increased macrophage infiltra-tion and increased expression of gal-3 and TNF-R.40

Acknowledgment. We thank Ann-Helen Thoren foranimal breeding and genotyping, Dr. Hakon Leffler, Dr. LarsHellman, Dr. John Wallace and Dr. Patrik Brundin for their kindgifts of antibodies. Financial support was provided by theSwedish Research Council (project no. 11284 to C.H. andproject no. 4499 to F.S.), Cell Factory for Functional Genomics,a program funded by the Swedish Foundation for StrategicResearch, Center of Excellence Grant from the Juvenile DiabetesFoundation, USA, and Knut and Alice Wallenberg Foundation,Sweden, the Swedish Diabetes Association and the followingfoundations: Novo Nordisk, Denmark, A. Påhlsson, Salubrin/Druvan and Torsten and Ragnar Soderberg.

Supporting Information Available: Evaluation of thedata analysis process in the 2D-PAGE experiment and twosupporting information tables (Tables S1 and S2). This materialis available free of charge via the Internet at http://pubs.acs.org.

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