J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

Profiling of gender-specific rat plasma proteins associated withsusceptibility or resistance to diet-induced obesity

Jung-Won Choi, Hao Liu, Duk Kwon Choi, Tae Seok Oh, Rajib Mukherjee, Jong Won Yun⁎

Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, Republic of Korea

A R T I C L E I N F O

Abbreviations: α1-III, alpha-1-inhibitor III;boxylesterase 1; Complement C, complemenprotein; FFA, free fatty acid; FGG, fibrinogencholesterol; HFD, high fat diet; Hp, haptoglobipeptide 2; ITIH, inter-alpha-trypsin-inhibitorretinol binding protein; SERPIN, serine (or cytransthyretin; TUBB5, tubulin beta-5 chain; U⁎ Corresponding author. Tel.: +82 53 850 6556

E-mail address: jwyun@daegu.ac.kr (J.W.

1874-3919/$ – see front matter © 2011 Elseviedoi:10.1016/j.jprot.2011.11.012

A B S T R A C T

Article history:Received 11 August 2011Accepted 12 November 2011Available online 20 November 2011

Obesity-prone (OP) and obesity-resistant (OR) rats with different responses to developmentof obesity in spite of the same genetic background are useful animal models for searchingfor markers during the development of obesity. Here, we investigated whether plasma pro-teins of OP and OR rats may behave in a different way in males and females. We performeda comparative proteomic analysis using 2-DE combined with MALDI-TOF/MS on proteinsfrom OP and OR male and female rats to discover gender-specific rat plasma proteins asso-ciated with susceptibility or resistance to diet-induced obesity. A total of 29 proteins show-ing differential expression between the groups were identified by MALDI-TOF/MS anddatabase searches. These proteins were classified into 4 groups according to their regula-tion patterns in response to diet and gender. 22 proteins showed significant differences be-tween OP and OR rats in males and/or females (Group I, II, and III) and 7 proteins exhibitedonly a high fat diet (HFD)-responsive difference in male or female rats (Group IV). In conclu-sion, the proteins negatively (ITIH3, FGG, TUBB5, and ZAG) or positively (Hp, ITIH4, and RBP)correlated with obesity found in this study could be used for selection of new targets forgender specific-medical treatment of obesity.

© 2011 Elsevier B.V. All rights reserved.

Keywords:2-DEHigh fat dietObesity-prone and -resistant ratsGender differencesObesityPlasma proteome

1. Introduction

Obesity is a heterogeneous complex disorder with multipleetiologies characterized by excess body fat that threatensmental or physical health [1]. Studying different responses toits development has become a critical research field [2]. Ro-dents are extensively used in such research and especiallythe diet-induced obesity (DIO) model provides an effectivesystem for developing the obese phenotype [3]. When outbredSprague–Dawley (SD) rats are exposed to a high fat diet (HFD),there is a wide distribution in body weight gain; some rats

A1M, alpha-1-macroglobut component; Cp, cerulopgamma chain; Ft, fetuin;n; Hpx, hemopexin; IGNTheavy chain; ND, normasteine) peptidase inhibitCP, uncoupling protein; V; fax: +82 53 850 6559.Yun).

r B.V. All rights reserved

easily become obese (obesity-prone; OP), whereas others re-main as lean as rats fed a normal diet (ND) (obesity resistant;OR) [4–6]. The physiological peculiarities of this model repre-sent a counterpart of human obesity with a polygenic modeof inheritance [7,8], a persistence of the phenotype [9], and adysregulated glucose homeostasis [6]. These features havemade this model useful for investigating obesity.

There are reports that OP and OR traits, namely regulationof caloric intake, pancreatic sympathetic tone, and plasma in-sulin levels, are transmitted in SD rats with a polygenic pat-tern of inheritance [7,8,10], and these differences may affect

lin; ACC, acetyl-CoA carboxylase; Apo, apolipoprotein; CES1, car-lasmin; CPT1B, carnitine palmitoyltransferase 1B; CRP, C-reactiveGSH-Px, glutathione peroxidase; HDL-C, high-density lipoprotein2, I-branching beta-1,6-acetylglucosaminyltransferase family poly-l diet; PRKA, protein kinase, AMP-activated, catalytic subunit; RBP,or; Tf, transferrin; TG, triglyceride; Total-C, total cholesterol; TTR,DBP, vitamin D-binding protein; ZAG, zinc-alpha-2-glycoprotein

.

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subsequent changes in diet composition and energy availabil-ity [6,11,12]. Moreover, animal blood and various tissues havebeen studied to discover markers associated with susceptibil-ity and resistance to obesity in rodents [3,13–15]. In our earlierproteomic studies, we identified numerous proteins showingdifferential regulation in plasma [16], adipose tissue [17], skel-etal muscle [18], and liver tissue [19] between OP and OR rats.

Obesity and its complications including various metabolicdisorders could be affected by gender. In humans, men andwomen get fat in different ways and can suffer different con-sequences [20]. These differences were expected to resultfrom differences between males and females in body fat dis-tribution [21–23], metabolic rate [24], oxidative capacity [25],and sex hormones [26,27]. Contradictory results have beenreported regarding differences in susceptibility to obesity be-tween males and females [28,29]. To date, differential proteo-mic studies on genders have analyzed blood [30] and varioustissues includingmuscle [31], kidney [32], and brain [33]. In ad-dition, gender specificity in plasma proteins has been dealtwith already a decade ago in rats under various conditionssuch as healthy state [34], acute-phase reaction [35], andstroke [36]. However, the role of gender in susceptibility andresistance to obesity is not entirely understood.

In our previous study, we performed profiling of plasma pro-teins in male SD rats fed a HFD and discovered several impor-tant proteins correlated with obesity [16]. Here, wehypothesized that proteins correlated with OP and OR traitsmay contribute differently in a gender-dependant manner. Wecarried out comparative proteomic analysis of plasma proteinsfrom OP and OR of male and female rats to discover gender-specific rat plasma proteins associated with susceptibility andresistance to diet-induced obesity. To the best of our knowl-edge, this is the first attempt to find gender-specific proteins as-sociated with development of diet-induced obesity.

2. Materials and methods

2.1. Animals and diets

Male (n=30) and female (n=30) SLC Sprague–Dawley (SD) ratswere purchased from Daehan Experiment Animals (Seoul,Korea) at 5 weeks of age, and housed singly in cages in a con-trolled environment (23±2 °C; 12:12-h light–dark cycle) withfree access to water and chow. After a 1 week adaption period,they were randomly assigned to either the normal (Feed KoreaLab, Hanam, Korea; 21, 68 and 12% calories as protein, carbo-hydrate and fat, respectively; males, n=10; females, n=10) orHF (20, 35 and 45% calories as protein, carbohydrate and fat,respectively; males, n=20; females, n=20) diet. The dietarycomposition of the ND and HFD is shown in SupplementaryTable 1. They were fed normal or HF diet for 8 weeks, andHFD rats were then subdivided into OP and OR rats accordingto their body weight gain. Some rats had body weight gain in-termediate between the two groups, and to improve the reli-ability of our proteomic study we excluded them when ratswere divided into OP (males, n=7; females, n=7) and ORgroups (males, n=7; females, n=7). All rats and feeds wereweighed every week for 8 weeks, and the animals were food

deprived for at least 12 h before sacrifice. Animal experimentswere approved by the Committee for Laboratory Animal Careand Use of Daegu University, and all procedures were con-ducted in accordance with the Guide for the Care and Use ofLaboratory Animals published by the National Institutes ofHealth.

2.2. Blood plasma sample and biochemical parameters

Blood was rapidly withdrawn into EDTA-tubes (BD, FranklinLakes, NJ, USA) from the end of the rat tail under anesthesiaby 3% diethyl ether. Blood samples were centrifuged(3000×g, 10 min) and plasma was collected, frozen in liquidnitrogen and stored at −80 °C. Protein concentration of plasmawas determined by the Bradford method [37] using proteinassay dye reagent concentrate (Bio-Rad, Hercules, CA, USA).Plasma biochemical parameters, including glucose, triglycer-ide (TG), total cholesterol (total-C), high-density lipoproteincholesterol (HDL-C), free fatty acid (FFA), leptin, insulin, andsex hormones (estrogen and testosterone) were measured in-dividually in triplicate using plasma of male and female ND-fed rats (n=7) and HFD-fed rats (n=7 for OP rats and n=7 forOR rats). Total-C and HDL-C were determined by enzymaticmeasurement using commercial kits form Bio Clinical SystemCorporation (Anyang, Korea), and TG and glucose were mea-sured by enzymatic kits from Asan Pharmaceutical (Seoul,Korea). FFAs were quantified using an enzymatic fatty acidquantification kit (Abcam, Cambridge, UK). Insulin and leptinlevels were gauged by a Rat Insulin kit (Millipore Co., Billerica,MA, USA) and Rat Leptin kit (Millipore), which are both sand-wich enzyme-linked immunosorbent assay (ELISA) systems.Sex hormone levels were measured using a Rat Estrogen ELISAkit (Cusabio Biotech. Co., LTD, Wuhan, Hubei, China) and RatTestosterone ELISA kit (Cusabio Biotech). Measurements wereperformed according to the manufacturer's instructions.

2.3. 2-DE analysis

2-DE experiments were conducted by the methods outlinedin our previous studies [16,38]. Briefly, whole plasma protein(25 μg) was applied to 18 cm IPG DryStrips, pH 4–7 (GEHealthcare, Buckinghamshire, UK). Strips were rehydratedwith protein in a solution which included 7 M urea (BioBasic, Ontario, Canada), 2 M thiourea (Sigma, St. Louis, MO,USA), 4% CHAPS (Bio Basic), 1 mM PMSF (Sigma), 20 mMDTT (GE Healthcare), and 2% IPG buffer (Bio-Rad) for 26 h.Then, immobilized pH gradient (IPG)-isoelectric focusing(IEF) was performed in a PROTEIN IEF cell (Bio-Rad) as fol-lows: 15 min at 250 V, 3 h at 250–10,000 V, 6 h at 10,000 Vand held at 500 V until samples were ready to run in the sec-ond dimension. Focused IPG strips were equilibrated twicefor 20 min with gentle shaking in equilibration buffer con-taining 6 M urea, 2% SDS (Generay Biotech, Shanghai,China), 1% DTT, 30% glycerol (Bio Basic) and 50 mM Tris–HCl (pH 6.8). In the second equilibration buffer, DTT wasreplaced with 2.5% iodoacetamide (Bio-Rad) in order to re-move excess DTT which causes point streaking in silverstained gels. The equilibrated strips were gently rinsed withdistilled water for removal of excess equilibration bufferand then applied onto a 12% polyacrylamide gel (20×20 cm)

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and fractionation was performed with the Laemmli SDS-discontinuous system, at a constant voltage of 15 mA pergel for 14 h. The gels with separated proteins were visualizedby the silver staining method for image analysis and peptidemass fingerprinting (PMF). 2-DE was carried out 3 timesusing the plasma of 7 rats per each group. These groups con-sisted of a control group which was fed a ND, as well as maleand female OP and OR groups which were fed a HFD.

2.4. Image analysis

Imaging gels produced by a UMAX PowerLook 1120 (MaxiumTechnologies, Akron, OH, USA)were analyzed bymodified Ima-geMaster 2-D software V4.95 (GE Healthcare). A reference gel se-lected from gels of the normal group was matched withindividual gels of OP and OR groups inmales and females. Rela-tive optical density and relative volumewere calculated for cor-rection for differences in gel staining. Each spot intensityvolume (%) was processed by background subtraction andtotal spot volume normalization; the resulting spot volume per-centage was used for comparison between groups.

2.5. Peptide mass fingerprinting (PMF)

Differentially regulated spots between groups were identifiedby PMF. Excised spots were digested with trypsin (Promega,Madison, WI, USA), mixed with α-cyano-4-hydroxycinnamicacid in 50% acetonitrile/0.1% trifluoroacetic acid. Peptideswere analyzed using matrix-assisted laser desorption/ioniza-tion-time-of-flight (MALDI-TOF; Ettan MALDI-TOF Pro, GEHealthcare). Spectra were collected from 350 shots per spec-trum over an m/z range of 600–3000, and calibrated by two

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Fig. 1 – Body weight profiles (A) and total body weight (B) of maleData are presented as mean±SD in 7 rats per group and statisticwere determined by a ANOVA test, where p value is *p<0.001.

point internal calibration using trypsin auto-digestion peaks(m/z 842.5099, 2211.1046). Peak lists were generated using theEttan MALDI-TOF Pro Evaluation Module (version 2.0.16). Thethreshold used for peak-picking was as follows: 5000 for mini-mum resolution of monoisotopic mass, 2.5 for S/N. The searchprogram MASCOT (Mascot Sever 2.3), developed by TheMatrixscience (http://www.matrixscience.com),wasused for pro-tein identification by PMF. The following parameters were usedfor the database search: trypsin as the cleaving enzyme, a maxi-mum of 1 missed cleavage, iodoacetamide (Cys) as a fixed modi-fication, oxidation (Met) as a variablemodification, monoisotopicmasses, and a mass tolerance of±0.1 Da. MASCOT probability-based MOWSE (molecular weight search) score was calculatedfor PMF. Protein score is −10⁎Log (p), where p is the probabilitythat the observed match is a random event, and scores >61were regarded as significant (p<0.05).

2.6. Immunoblot analysis

Levels of 6 proteins identified by 2-DE experiments were validat-ed together with 3 important plasma proteins by immunoblotanalysis. Plasma (70 μg) was diluted in 5× sample buffer (50 mMTris of pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol,and 0.1% bromophenol blue) and heated for 5min at 95 °C andthen SDS-polyacrylamide gel electrophoresis (PAGE) was con-ducted. Proteins separate by molecular weight were transferredto a polyvinylidene difluoride (PVDF, Santa Cruz Biotechnology,Santa Cruz, CA, USA) membrane and blocked for 1 h with TBS(Tris-buffered saline)-T buffer (10mM Tris–HCl, 150mM NaCl,and 0.1% Tween 20 containing 5% skim milk). The membranewas rinsed in 3 changes of TBS-T buffer, followed by incubationfor 1 h with a 1:1000 dilution of primary polyclonal antibody

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Fig. 2 – Plasma biochemical parameters in each experimental group. Levels of biochemical parameters were averaged in eachgroup and expressed as mean±SD of 7 separate experiments. Statistical significance between each group was determined byan ANOVA test, where p value is *p<0.05, **p<0.01, and ***p<0.001.

1389J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

(anti-Apo A-I, anti-Apo A-IV, anti-FGG, anti-Ft B, anti-VDBP,anti-ZAG, anti-adiponectin, anti-RBP4, anti-resistin or anti-β-actin; Santa Cruz Biotechnology) in TBS-T buffer including 1%skimmilk. After again washing 3 times, the membrane was in-cubated for 1 h with horseradish peroxidase-conjugated anti-goat IgG, anti-mouse IgG, or anti-rabbit IgG secondary antibody(1:1000, Santa Cruz Biotechnology) in TBS-T buffer containing1% skimmilk. Membranes were developed by enhanced chemi-luminescence (ECL; GE Healthcare) and identified bands werescanned by a UMAX PowerLook 1120 and digitalized usingimage analysis software (KODAK 1D, Eastman Kodak, Roches-ter, NY, USA).

2.7. Statistical analysis

Data are expressed as the mean±SD. Differences betweenthe groups were determined by one-way analysis of variance(ANOVA) using the Statistical Package of Social Science

(SPSS, version 14.0 K) program, followed by the protectedleast-significant difference (LSD) test for all pairwise multiplecomparisons. A p value <0.05 was considered statistically sig-nificant. In addition, Kolmogorov–Smirnov test for normalityinvestigation, Levene tests for examination of equal variance,and Tukey's test for post-hoc analysis were also performedusing SPSS software (Supplementary Table 2).

3. Results

3.1. HFD-induced phenotypes in male and female rats

Bodyweights of rats were similar betweenND and HFD groupsat the beginning of the study. However, after 2 weeks, HFDrats began to divide into OP and OR rats. At all subsequenttime points OP rats, both males and females, were heavier(p<0.001) than ND and OR rats, with a larger difference be-tween males (Fig. 1A). Total body weight gain was also

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Fig. 3 – A representative silver-stained 2-DE gel image of rat plasma proteome. Differentially regulated spots in each group aremarked with arrows, and the corresponding proteins are listed in Table 1.

1390 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

markedly higher in OP rats in both genders (Fig. 1B). Plasmabiochemical parameters, including glucose, TG, total-C, HDL-C, FFA, leptin, insulin, and sex hormones were measured inall groups. Results showed that glucose, TG, total-C, FFA, lep-tin, and insulin levels were significantly higher, whereasHDL-C was remarkably lower in OP rats, both males and fe-males, in comparison with ND and OR rats (Fig. 2). In particu-lar, plasma levels of glucose, leptin, and insulin were found tobe higher in males than in females (Fig. 2). The plasma levelsof estrogen were significantly higher in OP rats comparedwith ND and OR rats in both males and females. Conversely,plasma levels of testosterone were higher in ND and OR ratsthan in OP male rats (Fig. 2).

3.2. Observation of global protein regulation patternsat a glance

A total of 375 individual spots were detected in one gel fromour 2-DE-based proteomic experiments (Fig. 3). For comparingprotein regulation profiles globally in males vs. females or OPvs. OR groups, scatter plots were constructed using all spots(1125 individual spots) in three gels of each group (Fig. 4). Inorder to identify whether sex dimorphism in plasma proteinregulation pointed toward a higher tendency of male rats toundergo greater weight gain when exposed to HFD feeding,the plots were made in two different ways. One is to comparespot densities for males with those for females in OP and OR

groups (Fig. 4A), and the other is to make a comparison be-tween OP and OR rats in male and female groups (Fig. 4B). Agreater number of proteins differentially regulated by morethan 1.5-fold were observed for males than for females inthe OP group, and vice versa in the OR group. On the otherhand, among males more proteins were differentially regulat-ed in the OP group, among females in the OP group.

3.3. Proteins showing significant differences betweenOP and OR rats in both genders (Group I)

Among 375 individual spots, 29 proteins were found to bedifferentially regulated among groups; these were processedfor identification by MALDI-TOF/MS and database searches(Table 1). Sequences of the peptides identified by PMF for theplasma proteins differentially regulated in each group areshown in Supplementary Table 3. In order to reduce the possi-bility of false-positive hits, two representative spots (ITIH4and VDBP) were verified by CAF-MALDI-TOF/MS/MS sequenc-ing (Supplementary Fig. 1 and Supplementary Table 3). Theseproteins were classified into 4 groups according to their regu-lation patterns in response to diet and gender. Group I catego-rized proteins showing significant differences between OPand ND/OR rats in both males and females (Fig. 5); these in-cluded haptoglobin (Hp) inter-alpha-trypsin-inhibitor heavychain (ITIH) 4, retinol binding protein (RBP), ITIH3, fibrinogen

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Fig. 4 – Scatter plots comparing global protein regulation profiles betweenmales vs. females (A) or OP vs. OR rats (B). Regulationof plasma proteins of males or OP rats was plotted against those of the females or OR rats and these plots were drawn using allspots detected in three gels of each group. Spots with volume (%) of five and over were excluded from scatter plots. Upper andthe lower diagonal lines show the 1.5-fold regression lines. X- and Y-axes represent volume (%) of each spot in the normal, OP,and OR groups of males and females.

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gamma chain (FGG), tubulin beta-5 chain (TUBB5) and zinc-alpha-2-glycoprotein (ZAG). Hp, ITIH4, and RBP were up-regulated and FGG, TUBB5, and ZAG were down-regulated inOP rats compared with ND/OR rats in both genders, whereasITIH3 showed opposite regulation patterns between malesand females.

3.4. Proteins showing significant differences between OPand ND/OR rats in male (Group II) or female (Group III) rats

Group II and III categorized proteins showing significant differ-ences between OP and ND/OR rats in males or females, respec-tively (Fig. 6). Group II proteins consisted of ceruloplasmin(Cp), complement component 3 (C3), serine (or cysteine) pepti-dase inhibitor, clade G, member 1 (SERPING1), transferrin (Tf),vitamin D-binding protein (VDBP), fetuin A (Ft A), Ft B, glutathi-one peroxidase (GSH-Px), and hemopexin (Hpx). Ft A, Ft B, GSH-Px, andHpxwere decreased andCp, complement C3, SERPING1,Tf, and VDBP were increased in OP rats compared with ND/OR

rats only inmales. Group III proteins includedApo A-I, carboxy-lesterase 1 (CES1), alpha-1-macroglobulin (A1M), afamin, apoli-poprotein (Apo) A-IV, and C-reactive protein (CRP). Apo A-Iand CES1 were induced and A1M, afamin, ApoA-IV, and CRPwere reduced in OP rats compared with ND/OR rats only infemales.

3.5. Proteins showing only HFD-responsive differences inmale and/or female rats (Group IV)

Group IV categorized proteins showing only HFD-response inmales and/or females (Fig. 7). These consisted of transthyretintetramer (TTRt), alpha-1-inhibitor III (α1-III), ApoM, transthyr-etin monomer (TTRm), Apo E, I-branching beta-1,6-acetylglu-cosaminyltransferase family polypeptide 2 (IGNT2) andSERPINA3K. Only TTRt showed clear HFD-responsiveness inboth genders, whereas α1-III, Apo M, and TTRm exhibitedHFD-response only in males, and Apo E, IGNT2 and SERPI-NA3K showed HFD-response only in females (Fig. 7).

Table 1 – Differentially regulated plasma proteins in rats fed a normal diet and high fat diet between the genders.

Protein Acc. no. a Nominalmass(Mr) b

CalculatedpI

p values (males) p values (females)

ND OP ND ND OP ND

vs.OP

vs.OR

vs.OR

vs.OP

vs.OR

vs.OR

Group I; proteins showing significant changes between OP and ND/OR rats in both gendersHaptoglobin (Hp) gi:204655 39,101 6.27 0.026 0.032 >0.05 0.015 0.028 >0.05Inter-alpha inhibitor H4 heavy chain (ITIH4) gi:2292988 103,885 6.08 0.032 0.031 >0.05 0.042 0.049 >0.05Retinol-binding protein (RBP) gi:206589 20,046 5.67 0.034 0.021 >0.05 0.011 0.044 >0.05Inter-alpha inhibitor H3 heavy chain (ITIH3) gi:149034207 99,249 5.9 0.021 0.046 >0.05 0.0005 0.014 >0.05Fibrinogen gamma chain (FGG) gi:61098186 50,247 5.85 0.007 0.027 >0.05 0.002 0.002 >0.05Tubulin beta-5 chain (TUBB5) gi:7106439 50,095 4.78 0.017 0.017 >0.05 0.026 0.036 >0.05ZN-alpha-2-glycoprotein (ZAG) gi:620121 33,976 5.87 0.028 0.049 >0.05 0.014 0.006 >0.05

Group II; proteins showing significant changes between OP and ND/OR rats in male ratsCeruloplasmin (Cp) isoform CRA_a gi:149048530 121,387 5.39 0.01 0.047 >0.05 0.019 >0.05 >0.05Complement component 3 (C3) gi:158138561 187,746 6.06 0.018 0.003 >0.05 >0.05 >0.05 >0.05Serine (or cysteine) peptidase inhibitor, clade G,member 1 (SERPING1)

gi:40018558 55,804 5.54 0.035 0.047 >0.05 >0.05 >0.05 >0.05

Transferrin (Tf) gi:1854476 7853 6.94 0.034 0.016 >0.05 >0.05 >0.05 >0.05Vitamin D-binding protein (VDBP) gi:203941 55,079 5.76 0.041 0.02 >0.05 >0.05 >0.05 >0.05Fetuin A (Ft A) gi:231468 38,757 6.05 0.028 0.042 >0.05 0.04 >0.05 0.02Fetuin B (Ft B) isoform CRA_b gi:149019915 42,332 7.47 0.013 0.031 >0.05 0.032 >0.05 0.011Plasma glutathione peroxidase (GSH-Px) gi:6723180 25,653 8.26 0.042 0.012 >0.05 0.047 >0.05 >0.05Hemopexin (Hpx) gi:122065203 52,060 7.58 0.049 0.014 0.032 0.039 >0.05 >0.05

Group III; proteins showing significant changes between OP and ND/OR rats in female ratsApolipoprotein A-I (Apo A-I) gi:2145145 29,956 5.51 0.001 >0.05 0.003 0.012 0.016 >0.05Carboxylesterase 1 (CES1) gi:2506388 60,479 5.51 >0.05 >0.05 >0.05 0.04 0.042 0.049Alpha-1-macroglobulin (A1M) gi:81872093 168,388 6.46 0.006 >0.05 0.047 0.014 0.044 0.037Afamin, isoform CRA_a gi:149033757 70,190 5.87 >0.05 >0.05 >0.05 0.044 0.017 >0.05Apolipoprotein A-IV (Apo A-IV) gi:114008 44,429 5.12 0.004 >0.05 0.001 0.01 0.01 >0.05C-reactive protein (CRP) gi:8393197 25,737 4.89 0.01 >0.05 0.011 0.043 0.002 >0.05

Group IV; proteins showed showing only HFD-responsive difference in male and/or female ratsChain A, transthyretin tetramer (TTRt) gi:3212532 50,280 5.8 0.045 0.012 0.045 0.048 >0.05 >0.05Alpha-1-inhibitor III (α1-III) gi:83816939 165,038 5.7 0.041 >0.05 0.012 >0.05 >0.05 >0.05Apolipoprotein M (Apo M) gi:9506391 21,841 5.73 0.009 >0.05 0.006 >0.05 >0.05 >0.05Chain A, transthyretin monomer (TTRm) gi:3212532 13,122 6.04 0.01 >0.05 0.0004 >0.05 0.042 0.002Apolipoprotein E (Apo E) gi:37805241 35,798 5.23 >0.05 >0.05 >0.05 0.003 >0.05 0.001I-branching beta-1,6-acetylglucosaminyltransfer-ase family polypeptide 2 (IGNT2)

gi:40849882 46,022 7.66 >0.05 >0.05 >0.05 0.002 0.002 0.0005

Serine (or cysteine) peptidase inhibitor, clade A,member 3 K (SERPINA3K)

gi:133777069 45,719 5.24 >0.05 >0.05 >0.05 0.005 >0.05 0.009

a Acc. no. is a NCBInr database accession number.b The nominal mass is the integer mass of the most abundant naturally occurring stable isotope of an element. The nominal mass of amolecule is the sum of the nominal masses of the elements in its empirical formula.

1392 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

3.6. Validation by immunoblot analysis

To remove the possibility of technical errors and artifactual ef-fects in proteomic analysis, levels of 7 proteins of interest identi-fied fromproteomic experimentswere validated by immunoblot

Fig. 5 – Proteins showing significant changes between OP and Npresented asmean values±SD of volume density (%) of the changbetween each group was determined by an ANOVA test, where peach protein name, see abbreviations.

analyses. The immunological results fully support our proteomicdata, in that levels of FGG and ZAG were significantly decreasedin OP rats of bothmales and females (Group I), whereas Ft B andVDBP were markedly changed only in male OP rats (Group II).Plasma levels of Apo A-I, Apo A-IV and CRP were considerably

D/OR rats in males (M) and females (F) (Group I). Data areed spot in 3 individual gels from 7 rats. Statistical significancevalue is *p<0.05, **p<0.01, and ***p<0.001. For abbreviation of

1393J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

diversified only in female OP rats (Group III) when comparedwith ND and OR rats (Fig. 8). In addition, we investigated thelevels of 3 major plasma proteins related to various metabolic

Males Females

0.00

0.02

0.04

0.06

0.08

0.10

0.0

0.5

1.0

1.5

2.0 ITIH3

* * *** *

* *

*

0.00

0.02

0.04

0.06

0.08

0.10

0

1

2

3 ITIH4

* * * *

0.0

0.1

0.2

0.3

0.4 Hp

* *

* *

** **

*

0.0

0.4

0.8

1.2

1.6

Group I

FGG

** * ** **

* *

0.0

0.2

0.4

0.6

0.8

1.0 ZAG

* * * **

OP OR OP ORHFD HFD

OP

Males

RBP

* *

* *

Vo

lum

e %

TUBB5

* * * *

ND NDND

syndromes, including adiponectin, RBP4, and resistin, whichcould not be detected by a proteomic approach. Adiponectinand resistin showed only HFD-response, whereas RBP4 showed

OR OP ORHFD HFD

Females

ND

1394 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

markedly increased levels in OP rats compared with ND and ORrats in bothmales and femaleswithout significant difference be-tween the genders.

4. Discussion

Both men and women are susceptible to obesity, but the inci-dence and consequences differ between the genders due todifferences in the pattern of fat deposition, mobilization, utili-zation [20], metabolic rate [24], energy expenditure [39], andoxidative capacity [25]. Therefore, men have a more centralaccumulation of fat, whereas women have a more glutealand femoral accumulation [40]. These differences could be in-duced by differences in distinct hormonal effects between thegenders. Sex hormones in both males and females have beenclosely associated with the direct or indirect regulation of ad-iposity through physiological mechanisms [40]. In men, obesi-ty has usually been related to decreased plasma testosteronelevels and with increased estrogen concentration [41]. Inwomen, menopause-induced estrogen deficiency and in-creased androgenicity are related to increased abdominal obe-sity and a concomitant alteration in the metabolic risk profile[42]. Endocrine changes resulting from menopause, whichmeans a progressive loss of ovarian function, dramaticallymodi-fy women's levels of estrogen, progesterone, and inhibin [43].

Previous data have shown that males have a higher suscep-tibility to becoming obese, compared with females in bothhumans and rodents. For example, male mice are known to bemore likely to become obese than females, and ovariectomizedfemalemicemimicmalemice in susceptibility to obesity [28]. Inaddition, it was found that male SD rats gainmore weight thanfemales when they are fed the same HFD for 11 weeks [44,45].Recent research to investigate the prevalence of obesity inadults in Central Greece demonstrated that the rates of beingoverweight andobese in thepopulationof Thessaly are relative-ly higher inmales than in females [46]. These reports are in linewith our present results, in that male SD rats gain more weightthan female rats while consuming HFD for 8 weeks (Fig. 1A).However, some contradictory results also have been found inthe literature, whichmay be explained by differences of strains,age and nutritional state of the animals [29,39,47].

One of the prominent fields of obesity research is to deter-mine why one individual easily becomes obese and another re-sists in the presence of excessive food energy intake. When fedthe same diet, OR and OP rats significantly differ in metabolicefficiency andmodulation of food intake [18]. These phenotypicdifferences between OP and OR rats have been shown to be as-sociated with strain, diet, age, physiological signaling, respon-siveness, and metabolic processes [9,48,49]. Several studiesshowed that these differencesmay reflect changes in diet com-position and energy availability, including β-oxidation rate andfatty acid synthesis [6]. Moreover, oxidative capacity, glucoseuptake, thermogenic capacity, and mitochondrial activitycould also play roles in susceptibility to obesity in mice [50].However, the pathways involved in these phenotypic differ-ences are mostly unknown.

In our previous study, we discovered novel proteins posi-tively and/or negatively related to obesity using proteomicanalysis in various tissues [17–19] and plasma [16] of OP and

OR rats. Based on these findings, we hypothesized that pro-teins determining OP and OR traits may contribute differentlyin males and females. We therefore performed comparativeproteomic analysis in plasma from male and female OP andOR rats. Prior to proteomic analysis, we compared levels ofkey biochemical parameters including glucose, TG, total-C,HDL-C, FFA, leptin, insulin, and sex hormones from individualrats in each group. In lipid profiles, there were obvious differ-ences between OP and OR groups, whereas there was little dif-ference between males and females (Fig. 2).

In dogs, leptin and insulin are secreted into the blood in directcorrelationwith body fats and are transported into the brain [51],where they stimulate specific receptors on neurons in the hypo-thalamus and inducemetabolic responses including food intake,energy expenditure, and body weight [52]. In our study, plasmalevels of leptin and insulin are significantly higher in both maleand femaleOP rats in comparisonwithND/OR rats, in proportionto bodyweight (Fig. 2). Insulin and leptin are also sexually dimor-phic. Femaleshave decreased susceptibility to fatty acid-inducedinsulin resistance in humans [53], while males show a strongerphenotype than females for glucose intolerance, insulin resis-tance, and diabetes in mice [54]. In humans, leptin levels inblood are much higher in women than in men [55]. However,our results show that insulin and leptin levels are relativelyhigher in males than in females, because males have higherweight gain compared with females (Fig. 2). Therefore, these re-sults do not support the hypothesis of pivotal roles of leptinand insulin in gender dimorphism in obesity.

A contribution to gender differences in insulin sensitivitycould be due to the activity of sex hormones such as estrogenand testosterone. In a human study, decreases of estrogenand increases of testosterone levels are related with loss ofsubcutaneous fat, gain of visceral fat, and increase in insulinresistance [56]. Estrogen modulates energy balance and fatdistribution by interacting with leptin signaling pathways[57]. Leptin levels are inversely correlated with testosterone,and exposure of human fat cells to testosterone suppressesleptin expression in obese children and adolescents [58]. Con-sequently, leptin has a stimulatory effect on estrogen inwomen and a suppressive effect on testosterone in males, al-though other factors may be involved [59,60]. In this study, wefound a distinct gender difference in the levels of testosteroneand estrogen between OP and OR rats. By HFD, testosteronelevels in males are decreased by 50%, whereas estrogen levelsin females are increased by 30% only in the OP group (Fig. 2).Results of biochemical parameters demonstrate that thereare differences in levels of lipids, leptin, insulin, and sex hor-mones between OP and OR rats, in spite of an identical nutri-tional condition. In particular, leptin shows a markeddifference between males and females.

During proteomic analysis, we found various plasma pro-teins correlated with OP and OR phenotypes in a gender-dependent manner, and they were categorized into 4 groupsaccording to their regulation patterns in response to diet andgender. These groups include 7 proteins showing differencesbetween OP and ND/OR rats in both genders (Group I), 15 pro-teins showing differences between OP and ND/OR rats inmales (Group II) or females (Group III), as well as 7 proteinsshowing only HFD-response without gender differences(Group IV). Representative proteins in each group will be

Males Females

0.0

0.1

0.2

0.3

0.4

0.5 Complement C3

* ****

0.0

0.4

0.8

1.2

1.6 Cp

* * *

0.0

0.1

0.2

0.3

0.4

0.0

0.5

1.0

1.5

2.0

Group II

SERPING1

VDBP

Group III

0

1

2

3

4

5 Hpx *

* **

* *

* *

0

2

4

6

8

10 Tf

* *

*

* *

*

0.0

0.5

1.0

1.5

2.0

2.5

* *

Afamin*

0

1

2

3

**

** * *

Apo A-I*

**

0.0

0.1

0.2

0.3

0.4

0.5 Apo A-IV

** **

* *

*

**

0

1

2

3

4

5

* *

* *

* **

CRP

0

1

2

3

4

5 A1M * *

* * **

OP OR OP ORHFD HFD

OP OR OP ORHFD HFD

OP OR OP ORHFD HFD

0.00

0.05

0.10

0.15

0.20

0.25 GSH-Px

* * ** *

0

1

2

3

4 Ft A

* * **

*

*

0.0

0.4

0.8

1.2

1.6 Ft B

*** *

*

*

*

0

2

4

6 CES1

* * * *

Vo

lum

e %

ND ND ND ND ND ND

Fig. 6 – Proteins showing significant changes between OP and ND/OR rats only in male rats (Group II) and only in females(Group III). Data are presented as mean values±SD of volume density (%) of the changed spot in 3 individual gels from 7 rats.Statistical significance between each group was determined by an ANOVA test, where p value is *p<0.05 and **p<0.01. Forabbreviation of each protein name, see abbreviations.

1395J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

discussed here in the perspective of literature data connectingthem with obesity susceptibility and resistance.

We focusedon theup-regulation of ITIH4, andRBP, aswell asthe down-regulation of FGG, TUBB5, and ZAG in HFD-fed OPmale and female rats. Plasma levels of ITIH4, a type of proteaseinhibitor that plays a role in inflammation, wound healing, andcancer metastasis [61], were higher in HFD-fed OP rats com-pared to those of ND/OR rats. The role of ITIH in pathophysio-logical conditions has been extensively discussed, includingroles in acute ischemic stroke [62] and tumors [63]. However, ex-cept for our previous result [16], no report associates ITIH andobesity. Surprisingly, the ITIH isoform ITIH3 is also up-

regulated in HFD-fed OP male rats, whereas it is down-regulated in HFD-fed OP female rats (Fig. 5). These proteinswere previously unrecognized as being differentially regulatedin diet-induced obesity. However, the physiological significanceof this change remains to be determined.

We foundnogender differences in the levels of RBP,which is acarrier protein for retinol in the blood and acts as a signaling adi-pokine in humans [64]. It is well recognized that RBP provideslinks between insulin resistance, obesity, and type 2 diabetes[65,66] and the serum concentration of RBP4 is increased inobese subjects [65]. Our proteomic data also firmly establish RBPas a marker positively correlated with obesity in both genders.

Males Females

0.0

0.1

0.2

0.3

0.4 Apo E

**

**

* *

*

Group IV

HFD HFD

OP OR OP ORHFD HFD

0

2

4

6 α1-III

**

*

0.0

0.4

0.8

1.2

1.6 TTRm

**** **

***

0.0

0.2

0.4

0.6

0.8

1.0 TTRt

* *

**

*

*

0.0

0.4

0.8

1.2

1.6 IGNT2

***

** **

***

* *

0.0

0.5

1.0

1.5

2.0

2.5 SERPINA3K

** **

0.00

0.05

0.10

0.15

0.20 Apo M

** **

* *

**

OP OR OP ORHFD HFD

OP OR OP OR

Vo

lum

e %

ND ND

NDNDNDND

Fig. 7 – Proteins showing only HFD-response without gender differences (Group IV). Data are presented as mean values±SD ofvolume density (%) of the changed spot in 3 individual gels from 7 rats. Statistical significance between each group wasdetermined by an ANOVA test, where p value is *p<0.05, **p<0.01, and ***p<0.001. For abbreviation of each protein name, seeabbreviations.

1396 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

Interestingly, plasma levels of fibrinogen (FGG) are lower inHFD-fed obese rats (OP) of both genders than those in theirlean counterparts (ND and OR). This result is unexpected

Fig. 8 – Validation of differentially regulated plasma proteins in NLevels of 6 identified proteins from 2-DE analysis and 3 critical pl7 rats. For abbreviation of each protein name, see abbreviations.

when compared with earlier findings that recognized FGG asa hallmark of inflammation, cardiovascular disease, andother metabolic diseases in humans [67,68]. Additional

D, OP, and OR male and female rats by immunoblot analysis.asma proteins were investigated using 3 individual gels from

OP OR OP ORHp, ITIH4, RBP

FGG, TUBB5, ZAG

ITIH3

Cp, Complement C3, SERPING1,

Tf, VDBP

Ft A, Ft B, GSH-Px, Hpx

A1M, Afamin, Apo A-IV, CRP

Apo A-I, CES1

Fig. 9 – Summary of identified plasma proteins showing differential regulation in OP and OR rats of males and females. Forabbreviation of each protein name, see abbreviations.

1397J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

studies should be conducted to confirm and better under-stand the role of FGG and its molecular pathways in obesitysuggested by the present study.

One of the most remarkable outcomes of this proteomicstudy was finding lower plasma levels of tubulin beta-5 chain(TUBB5) in HFD-fed obese (OP) rats than in their lean counter-parts (ND and OR rats). TUBB5 belongs to β-tubulin family con-sisting of TUBB1, 2, 3, 4, and 6 and acts as an intracellularscaffolding protein [69]. A limited number of reports deal withthe relationship between TUBB5 expression and disease states.Bouwman et al. found that TUBB5 is down-regulated in adiposetissues after dietary intervention in a human population [70].Higher plasma levels of TUBB5 found in both male and femaleOR rats suggest its possible role in obesity resistance.

Our proteomic data also firmly establish that ZAG has asuppressive role in development of obesity in both genders.A wealth of information exists about the role of ZAG in obesi-ty. ZAG is a soluble protein with a lipid mobilizing effect. In-creased ZAG contributes to loss of body fat through itslipolytic effect [71], whereas decreased ZAG may induce lipidaccumulation in adipose tissue in Chinese subjects [72]. More-over, ZAG-knockout mice are susceptible to weight gain whenfed a HFD [73], and ZAG-overexpressing transgenic mice showdecreased body weight and fat [74]. Our data are in line withthese earlier findings in that higher plasma levels of ZAG inboth genders are detected in OR rats, identifying it as a potentindicator of obesity resistance in both males and females.

Of particular interest, it was found that regulation patternsof the fetuin family members in response to HFD were oppo-site between males and females. Fetuins are synthesized

mostly in the liver, and are expressed at high levels duringfetal life, primarily in blood and brain, although the changesof fetuin levels during development vary among species. Inaddition, fetuins are now considered key proteins in severalmetabolic pathways [75]. Ft A is an inhibitor of insulin recep-tor tyrosine kinase, and thus circulating Ft A is related to insu-lin resistance in humans [76]. It is also positively related to theamount of fat accumulation in the liver [77]. In our earlierstudy, we found lower plasma levels of FtA in male OP rats[16]. However, in this study, this was not the case for the fe-males, in that higher levels of FtA are observed in HFD-fedrat plasma (OP and OR rats). This result raises the possibilitythat plasma levels of FtA are gender-specific obesity markers.

The differential regulation of TTRs, which are transportproteins of thyroxine [78], is also an interesting finding. Wefound TTR in two aggregation states (TTRm, monomer formand TTRt, tetramer form), whose plasma levels were marked-ly different in males and females in response to HFD feeding.The plasma levels of TTRt were increased, whereas those ofTTRm were reduced upon HFD feeding in both genders. MaleOR rats showed the highest TTRt levels, whereas female ORrats exhibited the lowest levels of TTRm, suggesting thatTTR may play a role in obesity resistance.

In our previous study [16], we found that 12 plasma pro-teins showed their abundance differences between OP andOR male rats. When extended to gender-specific regulationpatterns in this study, we were able to find a total of 32 plasmaproteins that were significantly regulated. Of these, only twoproteins (ITIH4 and RBP) showed identical regulation patterns,whereas 8 proteins were found to be differently regulated

1398 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 3 8 6 – 1 4 0 0

between the genders. Other 22 proteins were identified for thefirst time in this study, and they were significantly regulatedbetween genders as well as OP and OR rats.

We also performed an immunoblot analysis to measurethe plasma levels of 3 proteins of metabolic importance (e.g.adiponectin, resistin, and RBP4), which were barely detectedon 2-DE proteinmaps. Adiponectin is an important adipocyto-kine with antidiabetic and antiatherogenic activities, whichare partly regulated through glucose and lipid metabolism[79], and anti-inflammatory effects [80]. Thus, reduction ofadiponectin levels is considered to be a major risk factor inthe pathogenesis of cardiovascular disease, type 2 diabetes,and other metabolic diseases [81]. Although HFD feeding re-duces plasma levels of adiponectin, OR rats tend to showhigher levels of adiponectin only in males and not in females.Conversely, levels of RBP4 and resistin are significantly in-creased upon HFD feeding in both genders, and OR males,but not OR females, are more resistant to such increases. Col-lectively, those 3 proteins possibly contribute to physiologicalfeatures of OP and OR rats, but mainly in male rats.

5. Conclusions

Through proteomic analysis using plasma of a HFD-induced ani-malmodel, we found 22 proteins thatwere differentially regulatedbetween OP and OR rats in males and females (Fig. 9). Althoughsome of these have already been linked with obesity, proteinswith identical or opposite regulation patterns according to genderare reported for the first time in this study. Although we did notclarifywhether sex-dimorphic regulationofplasmaproteinsmain-ly results fromhormonal effects, this couldbe firmlyestablishedbyusing orchiectomized and/or ovariectomized rodentmodels or sexhormone-treated cell lines in further studies. We expect that pro-teins potentially correlated with obesity identified in the currentstudy could provide the basis for research on the gender-dimorphic pathophysiological mechanisms underlying obesity.

Supplementary data to this article can be found online atdoi:10.1016/j.jprot.2011.11.012.

Acknowledgments

This research was supported by the Mid-career ResearcherProgram (grant number 2011-0000509) and SRC program (Cen-ter for Food & Nutritional Genomics: grant number 2011-0000914) of the National Research Foundation of Korea (NRF)funded by the Ministry of Education, Science and Technology.Jung-Won Choi, Hao Liu, Duk Kwon Choi, Tae Seok Oh, andRajib Mukherjee received the BK21 scholarship.

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