al

Wa

ine,

DIGE1H-NMR

anyanda rroue trs including DJ-1, Blvrb and AdoHycase were validated by Western blot.lites related to diseases such as lactate, fatty acid, glucose and homocysteine,

stress,al facto

well-being, as a risk factor for many diseases [

disease processes. In addition, inammation, infection, autoimmune to study liver responses at the proteomic and metabonomic levels

Biochimica et Biophysica Acta 1794 (2009) 17511765

Contents lists available at ScienceDirect

Biochimica et Bi

e lsprocesses, and perhaps even the onset and development of malignanttumors may occasionally be associated with the stress phenomenon[3]. Stress has also been shown to be important in vascular hyper-tension [4], and it may serve as a risk factor, induce blood pressurespikes, or increase an already elevated blood pressure [5,6]. Stressmay even cause or contribute to the clinical onset of liver disease incertain cases. In animals, the liver is one of the important organs,which plays many key roles in life processes, such as detoxication,metabolism of fat, protein and sugar, production of blood and bile etc.Some early clinical reports suggested that stress might affect the

during stress treatment. To our knowledge, there has been no previousreport about this process.

2-D DIGE (two-dimensional differential in-gel electrophoresis) is arecent improvement of the technique based on 2-DE. In 2-D DIGE,protein samples to be compared are labeled covalently with uorescentcyanine dyes, Cy2, Cy3, and Cy5, respectively. The labeled proteins aremixed, loaded, and separated in a single gel of 2-DE. Gel images withidentical protein spot patterns are acquired by scanning the gel atspecic wavelengths for each dye. Spot intensities can be compareddirectly between samples and quantied using image analysis software.initiation, course and outcome of liver diseaet al. revealed that emotional stress signicblood ow [7]. To animalmodels, Iwai et al. re

Corresponding authors. Tel.: +86 22 84655001; faxE-mail addresses: chenmingprotein@yahoo.com.cn (M

(L. Qian).1 These authors contributed equally to this work.

1570-9639/$ see front matter 2009 Elsevier B.V. Aldoi:10.1016/j.bbapap.2009.08.01219]. For instance, stresses and immune-related

disease. However, the biological changes in the liver during theprocess of stress remain largely unexplored. Therefore, we undertookplays a major role in immunological diseasMetabonomicsProteomics

1. Introduction

Numerous studies have establishedeffects of environmental or psychosociinjury occurs during restraint stress, including inhibition of glycolysis and gluconeogenesis in the liver, anddysfunction of fatty acid -oxidation. The results suggest a comprehensive map that addresses howfunctional proteins act on metabolites to produce energy and process materials in rat liver as it responds torestraint stress. Further functional study on these dynamic change proteins and metabolites may lead tobetter understanding of the mechanisms of stress-induced diseases.

2009 Elsevier B.V. All rights reserved.

a concept describing thers on physical or mental

shock stress exacerbated liver injury in rats treated with carbontetrachloride [8]. Further research showed that this stress paradigmcould aggravate -galactosylceramide-induced hepatitis [9]. Thus,growing evidences have shown that stress can have an effect on liverRestraint stressRat liver were observed to be increasing during 8 weeks of restraint stress. Our data indicated that subclinical hepaticKeywords: DIGE result, three proteinFurthermore, some metaboDynamic proteomic and metabonomic aninjury in rat liver during restraint stress

Ming Chen ,1, Yongqing Wang 1, Yun Zhao 1, LiqunZhihua Yang, Lingjia Qian Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medic

a b s t r a c ta r t i c l e i n f o

Article history:Received 28 April 2009Received in revised form 5 August 2009Accepted 6 August 2009Available online 18 August 2009

Stress is a risk factor for mcombined MALDI-TOF/TOFmetabonomic levels whenproteins and 32 chemical gimplicated in glycolysis, th

j ourna l homepage: www.ses. For example, Hiroseantly decreased hepaticported that electric foot-

: +86 22 23314818.. Chen), newjia@vip.sina.com

l rights reserved.ysis reveal dysfunction and subclinical

ng, Jingbo Gong, Lei Wu, Xiujie Gao,

1 Dali Road, Heping District, Tianjing 300050, PR China

diseases. In this study, we used uorescence difference gel electrophoresis1H-NMR to monitor the intracellular processes in rat liver at proteomic andat was treated with restraint stress for 8 weeks. Dynamic changes in 42ps were monitored and identied. These proteins and chemical groups wereicarboxylic acid cycle, fatty acid oxidation, and the urea cycle. To verify the

ophysica Acta

ev ie r.com/ locate /bbapapRecently, 2-D DIGE has been widely used, with high reproducibility andreliability, for identifying disease markers and proteins involved inbiological responses [10,11]. Metabonomics, another tool in systemsbiology, gives a window on the biochemistry of whole systems, andprovides crucial information on drug toxicity, disease processes, andgene and protein functions [12]. Therefore, proteomics and metabo-nomics provide powerful tools for global analysis and monitoring ofproteins and metabolites as they change during biological processes.

mode of the DeCyder (GE Healthcare) software package, followed by a

1752 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765However, data emerging from any single -omics approach can onlyprovide crude indications of gene or protein function. It has beenproposed that these limitations can be overcome by integrating dataobtained from two or more distinct approaches [13]. Such integrationshould not only improve functional annotations but also help toformulate biological hypotheses. Many recent reports have describedthe application of an integrated strategy in disease biomarker andtoxicology studies [1417].

In this paper, we have used a combination of proteomics andmetabonomics to study the biological process in rat liver duringrestraint stress. Rat liver proteins and metabolites were detectedusing 2-D DIGE and 1H-NMR, respectively. Our aimwas to monitor ratliver protein expression and metabolism to garner clues about themechanisms of stress-induced diseases.

2. Materials and methods

2.1. Reagents

Cy2, Cy3, and Cy5 were purchased from GE Healthcare (Uppsala,Sweden). DMF was purchased from Aldrich (Poole, Dorset, UK).Thiourea and urea were purchased from Fluka (Buchs, Switzerland).DTT, agarose, glycerol, bromophenol blue, CHAPS, mineral oil,acrylamide, Bis, Tris base, glycine, SDS, iodoacetamide, TEMED andimmobile DryStrip gels (24 cm, pH 310 NL) were purchased fromBio-Rad (Hercules, CA, USA). ACN andmethanol were purchased fromFisher (Fair Lawn, New Jersey, USA). TFA was purchased from Merck(Schuchardt, Hohenbrunn, Germany). SBD-F and TECP were pur-chased from Sigma (St. Louis, MO, USA). Protease inhibitor cocktailwas purchased from Roche (Penzberg, Germany). Sequencing gradetrypsin was from Promega (Madison, WI). All buffers were preparedwith Milli-Q water (Millipore, Bedford, MA, USA).

2.2. Experimental animal model

Thirty adult male Wistar rats weighing 200 g250 g were dividedrandomly into control and ve stress groups (1w, 2w, 4w, 6w, and8w), and ve rats were treated in each group. All rats were housed in apathogen-free environment at room temperature (2225 C) andmaintained on rat chow and tap water ad libitum before restraintstress. The restraint stress animal model was induced according to themethod of Galea et al [18], with slight modications. Briey,individual rats in the stressed group were placed in a specially builtsize-manipulable cabin for 6 h/day (from 9:00 am to 15:00 pm) for 1,2, 4, 6, and 8 weeks respectively. Control rats were not disturbedduring this period. The animal model experiments were repeated atleast three times. Rats were sacriced 24 h after the last day ofrestraint. Livers were rapidly excised, immersed in ice-cold PBS buffer,and washed three times. After washing, the livers were cut into largepieces, weighed, and minced into 1 mm32 mm3 pieces, and imme-diately frozen in liquid nitrogen. Blood samples were collected on icefrom rat heart into evacuated tubes containing EDTA as ananticoagulant. Plasma was separated within 30 min in a refrigeratedcentrifuge at 4 C, and stored at 80 C until analysis. All of theinvestigations conformed to the Guide for the Care and Use ofLaboratory Animals published by the US National Institutes of Health.The ethics committees of the Tianjing Institute of Hygiene andEnvironmental Medicine reviewed and approved our experimentalprocedures.

2.3. Samples for proteomic analysis

Rat liver samples were ground into a ne powder in liquidnitrogen and homogenized in lysis buffer (7 M urea, 2 M thiourea, 4%CHAPS, 30 mM Tris, pH 8.5, one complete proteinase inhibitor cocktail

tablet per 50 mL lysis buffer). For improved cell lysis, the solution wascomprehensive Biological Variance Analysis (BVA). Gel spots wereltered according to their t-test and 1-ANOVA values. The gel with themost spots was considered the master gel. Inter-gel matching wasperformed through the inclusion of the internal standard on each gel.More than 92% of the spots could bematched. Then, all of the eighteenimages were classied into six groups according to the experimentalcondition. The DeCyder BVA module was used for performingcomparative cross-gel statistical analyses of all spots, permitting thesonicated on ice for 1 min (with 1 s pulse-on and 2 s pulse-off toprevent overheating). The sample was incubated for 30 min at roomtemperature with repeated vortexing. Unbroken cells or connectivetissue were removed from the homogenate by centrifugation at25,000g for 30 min at 20 C. The supernatant was stored at80 C.Protein concentration was determined with the Bradford assay kit(Bio-Rad, Hercules, CA, USA) using albumin diluted in lysis buffer asstandard.

2.4. 2D-DIGE and imaging

Protein samples from the same treatment group were pooled for2D-DIGE. Then pooled samples were dissolved in lysis buffer (7 Murea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) to give stocksolutions with nal concentrations of about 5 mg/mL. Cyanine dyeswere reconstituted in 99.8% anhydrous DMF and added to labelingreactions at a ratio of 400 pmol Cy dye to 50 g protein in differentgroups following the cross-label rule, according to the manufacturer'sguidelines. The internal standard was created by pooling an aliquot ofall biological samples in the experiment and labeling it with one of theCy dyes (usually Cy2). Briey, 50 g of lysate was minimally labeledwith 400 pmol of Cy2, Cy3, and Cy5, respectively, and incubated on icefor at least 30 min in the dark. The labeling reaction was terminatedby adding 1 L of 10 mM lysine and incubating on ice for at least15 min in the dark. Two samples labeled with Cy3 and Cy5,respectively, were analyzed on the same gel, together with a pooledsample as an internal standard, labeled with Cy2. The DIGEexperimental design is shown in Table S1. Prior to IEF, differentiallylabeled samples to be separated in the same gel were mixed, andadded to an equal volume of 2 sample buffer (7 M urea, 2 M urea, 4%CHAPS, 130 mM DTT, 2% pharmalytes 310 NL), and nally 1 broughtto a total of 450 L with additional sample dissolved in rehydrationbuffer (8 M urea, 2% CHAPS, 0.5% pharmalytes 310 NL, 20 mM DTT).2-DE was performed with Amersham Biosciences (Uppsala, Sweden)IPGphor IEF and Ettan Dalt Twelve electrophoresis units. Pre-cast IPGstrips (24 cm, pH 310 NL) were used for the separation in the rstdimension with a total focusing time of 76 kVh at 15 C. Prior to SDS-PAGE, each strip was equilibrated with 10 mL equilibration buffer A(6 M urea, 50 mM TrisHCl, pH 8.8, 30% glycerol, 2% SDS, 10 mg/mLDTT) on a rocking table for 15 min, followed by 10 mL equilibrationbuffer B (6 M urea, 50 mM TrisHCl, pH 8.8, 30% glycerol, 2% SDS,25 mg/mL iodoacetamide) for another 15 min. The strips were thenloaded and run on 12.5% acrylamide gels. The running parameter wasset as a constant power of 15 mA per gel at 15 C for 1 h, 25 mA per gelat 15 C for 6 h, followed by 30 mA per gel at 15 C until thebromophenol blue dye front had run off the bottom of the gels.Labeled proteins were visualized by the Typhoon 9410 imager (GEHealthcare). All gels were scanned at 100 nm resolution, and theintensity was adjusted to insure that the maximum volume of eachimage was within 60,00090,000. Images were cropped to removeareas extraneous to the gel image using Image Quant V5.2 (AmershamBiosciences, UK) prior to analysis. Gel analysis was performed withDeCyder 6.5 (GE Healthcare), an analysis software platformdesigned specically for 2-D DIGE. Sets of gels were rst analyzed,and spots were counted, using the Differential in-gel Analysis (DIA)detection of differentially expressed spots between experimental

1753M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765groups (t-test and 1-ANOVA, p

identied in one spot, the single protein member with the highestprotein score was singled out from the multi-protein family.

All free induction decay (FID) data were Fourier transformed into1H-NMR spectra. All of the 1H-NMR spectra of liver tissue extractswere phase- and baseline-corrected and then data-reduced to equal

width (0.02 ppm), corresponding to the region of 9.40.2, using theVNMR 6.1C software package (Varian Inc, Palo Alto, CA). For the NMRspectra of aqueous soluble liver tissue extracts, the region of 5.14.7was removed to eliminate the artifacts of the residual waterresonance. For the aqueous liver extracts, the region 2.07 (residual

Blueeek

1754 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765Fig. 1. DIGE gel image of liver proteins from rat after 2 weeks and 6 weeks of stress. (A)from stressed rat at 6 weeks; (C) red (Cy5) image of proteins from stressed rat at 2 w

expression levels in the three samples. The spots having signicance (p

acetonitrile signal) was also removed from all spectra, while for thelipophilic extracts, the region 3.13.4 (residual methanol anddeuterated methanol) was removed from all spectra. Prior to PCA,all spectra data sets were imported into the SIMCA-P 10.0 softwarepackage (Umetrics AB, Umea, Sweden). Separately, these data weremean-centered and pareto-scaled. Pareto scaling gives each variablea variance numerically equal to its standard deviation. Score plotsfrom the rst two PCs were used to visualize the separation of groups,and the values of the PC loadings reected the NMR spectral regionsthat were altered as a function of stress time. Mean data werecalculated for each integrated region for each time point. Plots of PC1and PC2 on these data represent the metabolic changes with the timeof stress.

3. Results

3.1. Analysis of differentially expressed proteins

A goal of this study was to measure the changes in the rat liverproteome during restraint stress. For this purpose, 2-D DIGEexpression proles of rat livers were generated at 0-, 1-, 2-, 4-, 6-,and 8-week time points in the course of 8 weeks of restraint stress(Fig. 1). Up to 14761650 different spots (156060, n=6 Cy2images) were detected on six gels. DIGE analyses indicated 63 spotsthat exhibited statistically signicant dynamic expression changesacross all of six experimental time points (1-ANOVA, p

CH3, (CH2)n, CH2OCOR, CH=CH, CH3, CH3, tyrosine, C1H, uracil,5 GMP, nicotinurate), and 7 remained unchanged during stress,compared with the chemical groups levels of the control group. For

example, CH2COandCH2OPO2have several fold increases after 4 weeksof stress. Other molecules, such as bile acid and glycogen, decreased atleast 2-fold during the eight weeks of stress (Fig. 4D and Table S3).

tchehedint

1756 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765Fig. 2. DeCyder output showing several of the identied proteins. Enlarged regions of maof the individual spots are shown in the left panels. Graphical representations of all matcover time. Values are the standardized log of abundance. The two points at each time po

averaged values. The protein points labeled with Cy3 and Cy5 from the same gel are conned protein spots (blue circles) and the three-dimensional uorescence intensity prolesspots for a particular protein are shown in the right panel, with changes in expression

on the graph represent single values from different gels, and lines are plotted using the

cted with a broken line.

1757M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 175117653.4. Effect of restraint stress on the concentration of homocysteine in ratplasma

Fig. 5 shows the concentration of plasma homocysteine over stresstime. There was no obvious change in the stressed rats' homocysteineconcentration in the rst two weeks compared with control (3.040.65 mol/L vs. 2.620.47 mol/L, n=5, p>0.05). After 4 weeks ofrestraint stress, homocysteine concentration was increased by 2.5-fold, compared with the control group (7.861.37 mol/L vs. 2.670.51 mol/L, n=5). In groups stressed for 4, 6, and 8 weeks,homocysteine concentration was signicantly (p

Table 2Proteins identied by 2-D DIGE and MALDI-TOF-TOF.

Spotno.a

IPI no. Protein description Appearanceb 1-ANOVAp-value c

p-valueIDd

Mr(Da )

PI Coverage(%)e

Scoref Peptidehits g

Peptides identied

Lipid metabolism798 IPI00211225 Long-chain specic acyl-CoA

dehydrogenase18 (18) 0.0076 1.30E-10 47842 7.63 30% 145 2 K.AQDTAELFFEDVR.L

K.GFYYLMQELPQER.L1032 IPI00201413 3-ketoacyl-CoA thiolase,

mitochondrial18 (18) 2.6E-06 6.40E-47 41844 8.09 44% 508 5 K.DFTATDLTEFAAR.A

R.YALQSQQR.WR.VGVPTETGALTLNR.LK.VPPETIDSVIVGNVMQSSSDAAYLAR.HK.TNVSGGAIALGHPLGGSGSR.I

1416 IPI00207217 Enoyl-CoA hydratase 18 (18) 0.001 4.00E-38 31496 8.39 44% 420 5 K.NSSVGLIQLNRPK.AK.FLSHWDHITR.IK.SLAMEMVLTGDR.IK.LFYSTFATDDR.RK.AQFGQPEILLGTIPGAGGTQR.L

Carbohydrate metabolism1045 IPI00194045 Isocitrate dehydrogenase

[NADP]15 (18) 0.0042 1.00E-32 46705 6.53 51% 366 3 K.LILPYVELDLHSYDLGIENR.D

R.LVTGWVKPIIIGR.HK.GQETSTNPIASIFAWSR.G

998 IPI00231745 Fructose-1,6-bisphosphatase 1

18 (18) 0.032 5.00E-35 39584 5.54 69% 389 4 K.DFDPAINEYIQR.KK.SRPSLPLPQSR.AK.FPPDNSAPYGAR.YK.KFPPDNSAPYGAR.Y

1440 IPI00231767 Triosephosphate isomerase 18 (18) 0.0042 3.20E-30 26832 6.89 65% 341 3 K.FFVGGNWK.MK.DLGATWVVLGHSER.RK.LPADTEVVCAPPTAYIDFAR.Q

967 IPI00764333 Succinate coenzyme Aligase

18 (18) 0.015 8.00E-16 47116 6.64 47% 197 2 R.ETYLAILMDR.SK.INFDDNAEFR.Q

1353 IPI00194324 Pyruvate dehydrogenase E1component subunit beta

18(18) 0.0092 3.2E-11 38957 6.2 34% 151 2 R.IMEGPAFNFLDAPAVR.VK.TYYMSAGLQPVPIVFR.G

Protein metabolism452 IPI00471530 Cytosol aminopeptidase 18 (18) 0.011 6.4E-35 56115 6.77 57% 388 5 K.GVLFASGQNLAR.Q

K.DEIPYLR.KR.TLIEFLLR.FR.MPLFEHYTR.QR.LILADALCYAHTFNPK.V

1629 IPI00231757 Proteasome subunit alphatype-2

18 (18) 8.80E-05 3.20E-17 25910 6.92 53% 211 3 K.HIGLVYSGMGPDYR.VK.SILYDER.SK.LAQQYYLVYQEPIPTAQLVQR.V

716 IPI00421528 Proteasome 26S subunit,ATPase 3

15 (18) 0.014 2E-27 49518 5.09 50% 313 3 K.DSYLILETLPTEYDSR.VR.KIEFPMPNEEAR.AK.MNVSPDVNYEELAR.C

226 IPI00392830 77 kDa protein 12 (18) 0.01 0.03 76896 5.25 14% 61 1 K.HFSVEGQLEFR.A

Nucleobase\nucleoside\nucleotide and nucleic acid metabolism869 IPI00205018 Methylmalonate-

semialdehydedehydrogenase

15 (18) 2.60E-04 9.60E-06 57883 8.44 25% 96 1 K.AISFVGSNQAGEYIFER.G

1360 IPI00371243 Nicotinate-nucleotidepyrophosphorylase

18 (18) 0.018 5.00E-26 31277 5.98 57% 299 4 R.GPAHHLLLGER.VK.YGLLVGGAECHR.YK.LYAEGDIPVPHAR.RK.YGLLVGGAECHRYDLGGLVMVK.D

1504 IPI00555297 Adeninephosphoribosyltransferase

18 (18) 8.80E-04 5.00E-12 19751 5.78 92% 159 2 R.SFPDFPIPGVLFR.DR.DISPLLKDPDSFR.A

756 IPI00551812 ATP synthase subunit beta 15 (18) 0.012 5E-25 56318 5.19 46% 289 3 K.AHGGYSVFAGVGER.TR.VALTGLTVAEYFR.DK.VALVYGQMNEPPGAR.A

Amino acid and derivative metabolism214 IPI00207941 Dimethylglycine

dehydrogenase18 (18) 0.023 6.4E-66 95987 6.91 67% 698 5 R.IVNAAGFWAR.E

R.ISYTGELGWELYHR.RR.NITDELGVLGVAGPYAR.RR.LEEETGQVVGFHQPGSIR.LK.NYPATIIQEPLVLTEPTR.T

863 IPI00211127 Argininosuccinate synthase 18 (18) 0.0094 3.20E-06 46467 7.63 32% 101 2 R.MPEFYNR.FK.GQVYILGR.E

1034 IPI00210920 Aspartate aminotransferase 15 (18) 0.028 2.5E-22 47284 9.13 52% 262 3 R.FVTVQTISGTGALR.VR.DAGMQLQGYR.YK.ILIRPLYSNPPLNGAR.I

823 IPI00324633 Glutamate dehydrogenase 1 18 (18) 0.0074 2.5E-38 61377 8.05 34% 422 4 K.TFVVQGFGNVGLHSMR.YK.HGGTIPVVPTAEFQDR.IR.DSNYHLLMSVQESLER.KK.IIAEGANGPTTPEADKIFLER.N

462 IPI00475676 Delta-1-pyrroline-5-carboxylate dehydrogenase

18 (18) 9.70E-04 2.00E-08 61830 7.14 20% 123 1 K.VANEPILAFTQGSPER.D

1758 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765

Table 2 (continued)

Spotno.a

IPI no. Protein description Appearanceb 1-ANOVAp-value c

p-valueIDd

Mr(Da )

PI Coverage(%)e

Scoref Peptidehits g

Peptides identied

Response to stimulus376 IPI00191737 Serum albumin precursor 15 (18) 0.019 6.40E-07 68686 6.09 35% 108 1 K.LGEYGFQNAVLVR.Y477 IPI00231742 Catalase 18 (18) 0.0002 3.2E-52 59719 7.07 48% 561 5 R.GPLLVQDVVFTDEMAHFDR.E

K.NFTDVHPDYGAR.VR.LAQEDPDYGLR.DK.GAGAFGYFEVTHDITR.YR.LGPNYLQIPVNCPYR.A

1511 IPI00231107 Parathymosin 18 (18) 0.0093 0.00016 11552 4.15 44% 84 1 R.TAEEEDEADPKR.Q598 IPI00197770 Mitochondrial aldehyde

dehydrogenase15 (18) 0.027 2.5E-38 56453 6.63 50% 422 4 R.VVGNPFDSR.T

K.AAQAAFQLGSPWR.RK.TIPIDGDFFSYTR.HR.TFVQEDVYDEFVER.S

323 IPI00212622 Gastrin-binding protein 15 (18) 0.015 5.00E-08 82613 9.16 32% 119 3 K.TVLGVPEVLLGILPGAGGTQR.LK.NLNSEIDNILVNLR.LK.YESAYGTQFTPCQLLR.D

516 IPI00567316 60 kDa heat shock protein 18 (18) 0.037 1.3E-39 60917 5.91 48% 435 4 K.GANPVEIRR.GR.AAVEEGIVLGGGCALLR.CK.LVQDVANNTNEEAGDGTTTATVLAR.R.TVIIEQSWGSPK.V

169 IPI00471584 HSP90-beta 18 (18) 0.013 1.00E-13 83229 4.97 33% 176 5 K.IDIIPNPQER.TR.ALLFIPR.RR.RAPFDLFENK.KK.HFSVEGQLEFR.AR.GVVDSEDLPLNISR.E

1509 IPI00212523 DJ-1 18 (18) 4.60E-04 8.00E-53 19961 6.32 89% 567 5 K.GAEEMETVIPVDIMR.RK.MMNGSHYSYSESR.VK.GAEEMETVIPVDIMR.RR.AGIKVTVAGLAGKDPVQCSR.DK.GLIAAICAGPTALLAHEVGFGCK.V

Cell homeostasis235 IPI00196656 Ba1-667 18 (18) 0.026 5.00E-56 107379 8.35 34% 599 5 K.DCTGNFCLFR.S

R.LYLGHSYVTAIR.NR.KPVDQYEDCYLAR.IK.KPVTEFATCHLAQAPNHVVVSR.KK.LPEGTTYEEYLGAEYLQAVGNIR.K

626 IPI00365929 Protein disuldeisomerase A6 precursor

18 (18) 0.0015 2.50E-23 48730 5.05 33% 272 3 R.TGEAIVDAALSALR.QK.GSFSEQGINEFLR.ER.GSTAPVGGGSFPNITPR.E

Development1057 IPI00389571 Keratin, type II

cytoskeletal 818 (18) 0.0005 1.3e-042 53985 5.83 55% 465 5 K.LALDIEIATYR.K

R.LEGLTDEINFLR.QR.ATLEAAIADAEQRGELAVK.DR.AQYEEIANR.SR.LQAEIDALKGQR.A

Unclassied821 IPI00212104 Predicted 42 kDa

protein18 (18) 0.0032 8.00E-29 41942 5.77 52% 327 4 R.YEFEQQR.Y

R.VAAQAVEDVLNIAR.RR.ALNEVFIGESLSSR.AK.NVEHIIDSLRDEGIEVR.L

761 IPI00370348 28 kDa protein 15 (18) 0.04 2.00E-10 27731 4.71 20% 143 1 K.NQNINLENNLGEVEAR.Y949 IPI00193716 Isovaleryl-CoA.

dehydrogenase,mitochondrial precursor

18 (18) 0.024 1.00E-26 46406 8.03 42% 306 4 R.ASAAVGLSYGAHSNLCINQIVR.NR.AFNADFR.-R.QYVYNVAR.AK.GVYVLMSGLDLER.L

1630 IPI00364321 Electron transferavoprotein subunit beta

18 (18) 0.031 4.20E-06 27670 7.6 43% 100 1 R.GIHVEVPGAEAENLGPLQVAR.V

1489 IPI00392676 Predicted biliverdinreductase B

18 (18) 4.70E-05 4E-37 22083 6.29 78% 410 4 K.IAIFGATGR.TK.HDLGHFMLR.CR.LPSEGPQPAHVVVGDVLQAGDVDK.TK.YVAVMPPHIGDQPLTGAYTVTLDGR.G

824 IPI00476295 Adenosylhomocysteinase 18 (18) 0.0086 3.2E-06 47507 6.07 46% 101 1 K.SKFDNLYGCR.E1157 IPI00325765 Aatoxin B1 aldehyde

reductase member 218 (18) 0.01 1.30E-13 40649 8.35 43% 175 3 R.QVETELLPCLR.Y

R.FFGNSWSETYR.NR.WMYHHSQLQGTR.G

1487 IPI00205036 Hemoglobin alpha2 chain

15 (18) 0.0069 2E-45 15275 8.45 68% 493 4 K.IGGHGGEYGEEALQR.MK.LRVDPVNFK.FK.TYFSHIDVSPGSAQVK.AK.AADHVEDLPGALSTLSDLHAHK.L

992 IPI00212731 Cathepsin D precursor 18(18) 2.30E-05 0.0044 44652 6.66 16% 70 1 K.NIFSFYLNR.D1484 IPI00287835 Hemoglobin subunit

alpha-1/218 (18) 5.90E-04 1E-38 15319 7.82 71% 426 3 K.IGGHGGEYGEEALQR.M

K.TYFSHIDVSPGSAQVK.AK.AADHVEDLPGALSTLSDLHAHK.L

1759M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765

regulation of Blvrb was observed during the restraint stress timepoint, and AdoHycase levels increased and peaked at 4 weeks of stresstreatment, and then lowered, but are still above control levels at6 weeks of stress, before returning to approximately control levelsafter 8 weeks of stress. While DJ-1 protein was up-regulated duringthe rst week of stress treatment, and then down-regulated. It can beseen that the DJ-1 level down-regulated nearly 2-fold than control at4 weeks of stress treatment. These results were consistent with theDIGE results as shown in Supplemental data 1.

Notes to Table 2a Spot no. is the unique number of the position where the spot is displayed in the masteb Appearance means the number of gel maps in which one certain spot could be detectec p-value of 1-ANOVA test.d p-value ID is the best expectation value of identied protein as calculated by Mascot.e Amino acid sequence coverage for the identied protein.f Score is the protein score based on combined mass and mass/mass spectrums.g Peptide hits is the unique number of MS/MS spectrums which match to the trypsin pe

Fig. 3. Gene ontology annotation of identied proteins. Identied proteins were sub-mitted for gene ontology analysis using GOfact, a freely available web-based programfrom the laboratory of systems biology at the Beijing Institute of Radiation Medicine(http://www.hupo.org.cn). Identied proteins were categorized based on their(A) subcellular locations; (B) biological processes; and (C) molecular functions. Pie-chart values represent the number of proteins found in the given category for allsubmitted proteins with GO annotations.

1760 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 175117654. Discussion

4.1. Stress responsive proteins

Expression levels of eight identied proteins, including aldehydedehydrogenase (ALDH), parathymosin (ParaT), DJ-1, gastrin-bindingprotein (GBP), catalase, serum albumin precursor, Hsp60, and Hsp90correlated to the stress response (Table 2). ALDH is a group ofenzymes that catalyze the oxidation of aliphatic and aromaticaldehydes to the corresponding acids via a pyridine nucleotidedependent reaction. ALDH was dened as a stress response proteinby Han et al, who reported that ALDH was up-regulated in E. coliunder four different types of stress [21]. In our study, we found thatALDHwas down-regulated during the rst 4 weeks of stress, and thenup-regulated (Supplemental data 1).

We observed that ParaT was down-regulated during the process ofrestraint stress (Supplemental data 1). ParaT is expressed in all celltypes and is widely distributed in mammalian tissues, with thehighest concentrations found in the liver, heart, and kidney [22,23].Early studies identied ParaT as a zinc-binding protein that interactswith several enzymes involved in carbohydrate metabolism [24,25].In other reports by Okamoto and Isohashi, ParaT was found to inhibitthe binding of the activated glucocorticoid receptor to nuclei,suggesting its involvement in the regulation of glucocorticoid action[26,27]. We also observed an increase in glucocorticoid (GC) instressed rat liver (data not shown). Therefore, our results suggest thatParaTmight participate in regulation of the GC pathway, which plays akey role in the process of restraint stress.

In our study, we found that DJ-1 was up-regulated during the rst2 weeks of stress, and then down-regulated (Supplemental data 1).This down-regulation was further validated by Western blot (Fig. 6).DJ-1, originally identied as an oncogene product, is a protein withvarious functions in cellular transformation, oxidative stress response(mitochondrial) and transcriptional regulation, and its oxidative stateat cysteine residues determines activities of DJ-1. Fan et al. reportedthat DJ-1 exerts its cytoprotection through inhibiting the p53-Bax-caspase pathway. Over-expression of DJ-1 decreases the expression ofBax and inhibits caspase activation, whereas knockdown of DJ-1increases Bax protein levels and accelerates caspase-3 activation andcell death induced by UV exposure [28]. Mo et al. reported that DJ-1protects against UV-induced cell death also through the suppressionof the MEKK1-SEK1-JNK1 signaling pathway [29]. Many publicationshave reported that heat shock proteins are induced by stress [3032].Two heat shock proteins, Hsp60 and Hsp90, show decreasingexpression during the rst 4 weeks of stress treatment, and thenincrease (Fig. 2 and Supplemental data 1). Hsp60 is a mitochondrial

r gel.d (there were 18 gel maps in total in our study).ptide.

1761M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765chaperone, typically held responsible for refolding and transportingproteins from the cytoplasm into the mitochondrial matrix. Inaddition to its role as a heat shock protein, Hsp60 plays an importantrole in the transport and maintenance of mitochondrial proteins, andthe transmission and replication of mitochondrial DNA [31,32].Therefore, we think that the dramatic change in DJ-1 and heatshock proteins in rat liver may relate to the dysfunction ofmitochondria under stress conditions.

4.2. Homocysteine accumulated in restraint stressed rat plasma

Homocysteine is a sulfur amino acid and a normal intermediate inthe methionine metabolism pathway. This pathway involves theformation of S-adenosylmethionine (SAM), which subsequentlytransfers a methyl group to methyl acceptor molecules, and formsadenosylhomocysteine, which is subsequently converted to homo-cysteine. Homocysteine is then either converted back to methionineby remethylation, or further metabolized to cysteine via the trans-sulfuration pathway. When excess homocysteine is produced in thebody and not readily converted into methionine or cysteine, it isexcreted from the tightly regulated intracellular environment into the

Fig. 4. Effect of restraint stress onmetabolism in rat liver. (A) Principle Component Analysis sat (red ) 0 weeks; (green ) 1 week; (blue ) 2 weeks; (yellow ) 4 weeks; (purple )6liver metabolism under stress conditions over time. (B) 600 MHz 1H NMR spectra in low chemof lipid soluble liver tissue extract in stressed rats. Metabolites were assigned according to th8 weeks of restraint stress (more information can be seen in Table S3).blood as cytotoxin. It is the role of the liver to remove excesshomocysteine from the blood. In many individuals with liver disease,homocysteine levels can rise beyond normal levels (i.e. hyperhomo-cysteinemia) and lead to adverse health outcomes [33,34]. Usually,homocysteine in rat plasma was diet-induced, i.e. by feeding high-methionine diet [35,36]. However, it has been proved that restraintstress has signicant inuence on the adrenal cortex and then lead tovasculature and metabolism system damage in rats [3739]. Further-more, de Souza et al. and de Oliveira et al. reported their results after arat was treated using different stress manipulations, includingrestraint stress, swimming and cold, and found that not all stressmanipulations could increase homocysteine concentrations in ratplasma, and restraint stress was the only type of stress that alteredhomocysteine concentrations in rat [40,41]. So, their papers indicatethe specic relationship between restraint stress and the increase ofhomocysteine levels. In our study, we can see that homocysteineconcentration was increased more than 3-fold after 8 weeks ofrestraint stress (seen Fig. 5). These results indicate that restraintstress may lead to homocysteine accumulation in rat plasma.

Two proteins related to homocysteine metabolism were identiedin rat liver. Adenosylhomocysteinase (AdoHycase, SAHH) is an enzyme

cores plot from 1H NMR spectra of aqueous soluble liver tissue extracts from stressed ratweeks; and (black) 8 weeks. This plot emphasizes the large degree of variation in ratical shift regions (05.5) of water-soluble liver tissue extracts and (C) 1H NMR spectrae chemical shift value. (D) The dynamic change of partial metabolites in rat liver during

that catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHyc) toform adenosine and homocysteine. In recent years, elevated homo-cysteine was considered an independent risk factor for many diseases,including cardiovascular disease, diabetes, gastric ulcer, cancer, andParkinson's disease [42,43]. In the present study, we found thatAdoHycase was up-regulated during the rst 4 weeks of stress, and

then down-regulated (Fig. 6). Another protein correlated with homo-cysteine metabolism is biliverdin reductase (Blvrb), which is a serine/threonine kinase that catalyzes the reduction of biliverdin to producebilirubin. Domain analysis shows that this protein has an AdoHycasesuper-family domain (data not shown). In contrast to AdoHycase, Blvrbwas up-regulated continuously (Fig. 6). We noted that the weeklyincrease in homocysteine levels does not correlate with the AdoHycaseWestern blot data (Fig. 6) which shows that AdoHycase levels peak atweek 4, and return to approximately control levels at week 8, whereasthe homocysteine level increases throughout the 8 weeks of stresstreatment (Fig. 5). This may be due, in part, to cumulative build up ofhomocysteine. On the other hand, we suppose that Blvrb might involvein homocysteine metabolism.

It is reported that homocysteine can be induced by smoking,alcohol consumption, low consumption of fruits and vegetables, highintake of methionine-containing proteins, cystathionine -synthase(CBS) or/and methionine synthase deciencies [4446]. Our dataindicated that homocysteine can be induced by restraint stress(Fig. 5). For many years, the nutritional disorder or deciency ofCBS in hyperhomocysteinemia has been the focus of research [4749].Our results show that homocysteine can be elevated along with theup-regulation of AdoHycase and Blvrb under stress conditions.

4.3. Carbohydrate metabolism in rat liver during restraint stress

In this study, we identied 4 proteins that are related tocarbohydrate metabolism, including fructose-1,6-bisphosphatase 1(Fbp1), triosephosphate isomerase (Tpi1), isocitrate dehydrogenase(Idh) and pyruvate dehydrogenase E1(Pdhb). Fbp1 catalyzes hydro-lysis of fructose-1,6-diphosphate to fructose-6-phosphate, which isthe reverse reaction of rate-determining step in glycolysis [17]. The

Fig. 5. Effect of restraint stress on homocysteine concentration in rat plasma. Homocysteine(Hcy)wasmeasured by high-performance liquid chromatography. Chromatogramof plasmasample from rat stressed for 0, 2, 4, 6 and 8 weeks; Data are presented as meanSEM, andanalyzed by t-test.

1762 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765Fig. 6. Western blot validation of 3 proteins. Western blot of AdoHycase (Left top), Blvrb (normalized by -actin based on OD value of protein bands. Graphical representations of AdoHpanel with changes in expression over time. The result from the Western blot was consisteLeft middle) and DJ-1, -actin as an internal standard. The change ratio of proteins isycase (Right top), Blvrb (Right middle) and DJ-1 (Right bottom) are shown in the right

nt with the DIGE result (generated by DeCyder, see Supplemental data 1).

up-regulation of Fbp1might inhibit glycolysis. Tpi1, the other enzymeinvolved in the glycolysis, is also up-regulated. Interestingly, thisenzyme converts dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. Of the two, only glyceraldehyde 3-phosphate can takeparticipation in glycolysis. Therefore, we suggest that the increasedexpression of Tpi1 is a remedy for inhibited glycolysis in rat liverunder stress. All in all, glycolysis was partially inhibited under thestress of our experiments. Evidence for this result is also provided bythe increase in liver glucose and decrease in glycogen levels detectedby 1H-NMR (Fig. 4D and Table S3). Lactate, a potential source ofendogenous molecular toxins, is the end product of glycolysis [50].Jing et al. reported that increased lactate is a key biomarker related tolipodystrophy [51]. Higher levels of lactate were observed in stressedrat liver compared to control in our study (Fig. 4D and Table S3). Theincrease in the lactate level also suggested that gluconeogenesis wasinhibited, and changes in carbohydrate and energy metabolismoccurred. The effects of various stresses have been well documentedin the rat, including diet/water deprivation, heat shock, sleepdeprivation and acclimatization [5255]. It has been reported thatperturbations of liver metabolism by liver toxins can increase the rateof glycogenolysis [56,57]. Combining these results with our own, wespeculate that inhibition of glycolysis and gluconeogenesis in livermay be a response common to the various stress conditions.

We also observed a distinct change in TCA-related enzymes(isocitrate dehydrogenase and pyruvate dehydrogenase E1). Pyruvatedehydrogenase E1 is a key entry point for carbon in the TCA cycle.Isocitrate dehydrogenase converts isocitrate to oxalosuccinate and -ketoglutarate during the TCA cycle. It has been reported that increasedstress load is connected to an increased energy requirement, and leadsto metabolic readjustment. Hoffmann et al. reported that heat shock

stress can induce TCA cycle-related enzymes in the E. coli expressionsystem [58]. In our study, we found that isocitrate dehydrogenase andpyruvate dehydrogenase E1 (two key enzymes in TCA) show dramaticchanges. These two enzymes increased continuously during the rst4 weeks of stress, and then decreased (Supplemental data 1). Supportfor this result is provided by the same tendency of succinate andacetate in stressed rat liver at the metabonomic level (Fig. 4D andTable S3). Kil et al. reported that the primary function of mitochon-drial NADP+ dependent isocitrate dehydrogenase is the control ofcellular defenses against oxidative damage through the supply ofNADPH, which is needed for the generation of glutathione [59].Although glutathione was not detected in our 1H-NMR analysis, therelationship between stress and oxidative damage has been reported.So, we can propose that the TCA cycle may be activated in the earlyresponse to stress conditions, due to requirements for energy andredox balance.

4.4. Lipid metabolism in rat liver during restraint stress

DIGE analyses suggested that expression of three pivotal proteinsinvolved in fatty acid -oxidation were affected by stress treatment,including long-chain specic acyl-CoA dehydrogenase (Acadl), 3-ketoacyl-CoA thiolase (Acaa2, mitochondrial) and enoyl-CoA hydra-tase (Echs1). Acadl, the enzyme that catalyzes the rst reaction in the-oxidation of fatty acids, had a tendency to decrease during the stressperiod (Supplemental data 1). Another enzyme, Acaa2, whichconverts -ketoacyl-CoA to acetyl CoA, was also down-regulated. Onthe other hand, 1H-NMR analyses indicated that the levels of saturatedfatty acyl (Lipid CH2CO) and unsaturated fatty acyl (Lipid CH2C=C)were elevated. Triglycerides, considered as storage for excess fatty

sse

1763M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765Fig. 7. The summarized dynamic change at proteomic and metabonomic levels in stre

decreasing concentration in the course of 8 weeks of stress, respectively. Metabolites and pd rat liver cell. Metabolites and proteins in red and blue represent an increasing and

roteins in black means they were not detected in our experiment.

1764 M. Chen et al. / Biochimica et Biophysica Acta 1794 (2009) 17511765acid in the liver, were decreased (Table S3). This suggests that fattyacid was accumulated in the liver. Saturated and unsaturated fattyacids differ signicantly in their contributions to lipotoxicity. Previousstudies in Chinese hamster ovary cells, cardiac myocytes, breastcancer cell lines, and hematopoietic precursor cell lines all suggestedthat lipotoxicity from the accumulation of long-chain fatty acids isspecic to, or made much worse by, saturated fatty acids [6064].These data predict that increased hepatic saturated fatty acids maypromote liver damage in stressed rat, and perturbation in fatty acidoxidation and metabolic systems may decrease energy usage in theliver, leading to lipid storage in the liver cells.

Bile acids serve many important physiological functions, includingcholesterol homeostasis, lipid absorption, and generation of bile ow,all of which help to excrete and recirculate drugs, vitamins, andendogenous and exogenous toxins [65]. Many diseases, such ashepatobiliary and intestinal diseases, will affect the concentration ofbile acids. Therefore, bile acid has long been considered to be a markerfor liver injury [66]. In our study, we found that bile acid in liver tissuewas decreased by 1.5- to 6-fold during the 8 weeks of stress treatment(Fig. 4D and Table S3). This is in contrast with the ndings in a study ofhuman hepatocellular carcinoma, where an increase in the level ofbile acid was observed [51]. The authors propose that the increaseoccurred with bile duct obstruction, which is common in liverimpairments caused by propagation and invasion of hepatocellularcarcinoma (HCC) cells in the bile duct. In our study, we suggest thatlower levels of bile acids in liver tissue indicate that a subclinicalhepatic injury may be occurring.

In summary, using -omics strategies, we found that a combina-tion of information from protein and metabolite levels provides anintegrated picture of the response to restraint stress in rat liver. Weidentied 42 proteins and 32 chemical groups, implicated inglycolysis, TCA cycle, fatty acid oxidation, and the urea cycle. Ourdata suggests that subclinical hepatic injury may be occurring,including inhibition of glycolysis and gluconeogenesis, and dysfunc-tion of fatty acid -oxidation. Homocysteine, a putative risk factor ofmany diseases, could be elevated by increased adenosylhomocystei-nase and biliverdin reductase activity during stress. Our datacomprehensively mapped rat liver responses to restraint stress,showing changes in the interactions of proteins and metabolitesthat produce energy and process materials in liver cell (summarizedin Fig. 7).

Acknowledgements

The authors thankWenfeng Yu, Yujuan Yan and Xianzhong Yan fortheir assistance with the DIGE and 1H-NMR experiment. We alsothank two anonymous reviewers for their patient review and criticalreading of our manuscript. This research was supported in part bygrants from the National High Technologies R&D Program of China(2006AA02Z4C08) and the Key Project for Application and BasicResearch of Tianjing Municipality (06YFZJC02400).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbapap.2009.08.012.

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Dynamic proteomic and metabonomic analysis reveal dysfunction and subclinical injury in rat liv.....IntroductionMaterials and methodsReagentsExperimental animal modelSamples for proteomic analysis2D-DIGE and imagingIn-gel digestionMass spectrometer analysisWestern blot1H-NMR spectroscopy of water-soluble and lipid soluble liver tissue extractsMeasurement of homocysteine concentration in rat plasmaData analysis

ResultsAnalysis of differentially expressed proteinsIdentification of differentially expressed proteinsAnalysis of differential metabolitesEffect of restraint stress on the concentration of homocysteine in rat plasmaProtein validation by Western blot

DiscussionStress responsive proteinsHomocysteine accumulated in restraint stressed rat plasmaCarbohydrate metabolism in rat liver during restraint stressLipid metabolism in rat liver during restraint stress

AcknowledgementsSupplementary dataReferences