Proteomic Analysis of the Intestinal Adaptation Response Reveals Altered Expression of Fatty Acid...

Proteomic Analysis of the Intestinal Adaptation Response Reveals

Altered Expression of Fatty Acid Binding Proteins Following Massive

Small Bowel Resection

Andrew N. Stephens,*,†,# Prue M. Pereira-Fantini,*,‡,# Guineva Wilson,§ Russell G. Taylor,§

Adam Rainczuk,† Katie L. Meehan,† Magdy Sourial,§ Peter J. Fuller,† Peter G. Stanton,†

David M. Robertson,† and Julie E. Bines‡,|,⊥

Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Murdoch Children’s ResearchInstitute, Departments of Surgery and Gastroenterology and Clinical Nutrition, Royal Children’s Hospital, and

Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia

Received October 28, 2009

Intestinal adaptation in response to the loss of the small intestine is essential to restore enteral autonomyin patients who have undergone massive small bowel resection (MSBR). In a proportion of patients,intestinal function is not restored, resulting in chronic intestinal failure (IF). Early referral of such patientsfor transplant provides the best prognosis; however, the molecular mechanisms underlying intestinaladaptation remain elusive and there is currently no convenient marker to predict whether patients willdevelop IF. We have investigated the adaptation response in a well-characterized porcine model ofintestinal adaptation. 2D DIGE analysis of ileal epithelium from piglets recovering from massive smallbowel resection (MSBR) identified over 60 proteins that changed specifically in MSBR animals relativeto nonoperational or sham-operated controls. Three fatty acid binding proteins (L-FABP, FABP-6, andI-FABP) showed changes in MSBR animals. The expression changes and localization of each FABPwere validated by immunoblotting and immunohistochemical analysis. FABP expression changes inMSBR animals occurred concurrently with altered triglyceride and bile acid metabolism as well as weightgain. The observed FABP expression changes in the ileal epithelium occur as part of the intestinaladaptation response and could provide a clinically useful marker to evaluate adaptation following MSBR.

Keywords: 2D-PAGE • DIGE • intestinal failure • MSBR • adaptation • short bowel syndrome • fatty acidbinding protein • FABPL • FABP6 • FABPI

Introduction

Short bowel syndrome (SBS) is a state of malabsorption andmalnutrition, arising from either congenital disease or massivesmall bowel resection (MSBR), that results in a significant lossof functional intestinal length.1-3 In newborns and infants, themortality associated with SBS can reach 90%, making it one ofthe most lethal conditions in infancy and early childhood.3-9

In a small number of SBS patients, the remaining functionalbowel is unable to supply sufficient energy and nutritionrequirements to maintain growthsa state known as intestinalfailure (IF)sand the patient becomes dependent on long-term

parenteral nutrition (PN) for survival.2,10 However, complica-tions arising from the chronic administration of PN, such asbacterial sepsis and parenteral nutrition-associated liver disease(PNALD), are implicated in up to 5% of annual, nondiseaserelated mortality and make long-term PN undesirable.2,3,7,11-13

Chronic PN also significantly alters quality of life and has highassociated health-care costs.3,14 Bowel transplantation has beenproposed as a preferred treatment for patients with IF,14-18

but the long-term requirements for immunosuppression coupledwith a 5-year survival rate similar to chronic PN currently maketransplantation unsuitable for most IF patients.3,15,16,19

Following a significant loss of mucosal surface after MSBR,the remaining intestine must undergo compensatory physi-ological and morphological changes to increase its absorptivecapacity.10,20 This process of intestinal adaptation, involvingcompensatory growth and the dilation, thickening, and length-ening of remaining intestine, can be detected almost im-mediately and may last for a period of months to years.3,10,20,21

The adapting intestine undergoes prominent hyperplasia andhypertrophy in all layers of the bowel wall, a process primarilydriven by intestinal epithelial stem cells within the intestinalcrypts.22 Increased epithelial cell proliferation and migrationalong the crypt-villus axis results in lengthening of the villi,

* To whom correspondence should be addressed. Dr. Andrew N. Stephens,Level 4, Prince Henry’s Institute of Medical Research, PO Box 5152, ClaytonVIC 3168, Australia. Phone +613 95947912. Fax +613 9594 7909. [email protected]. Dr. Prue Pereira-Fantini, MurdochChildren’s Research Institute, Parkville VIC 3052, Australia. Phone +613 8341-6452. Fax +613 8341-6449. E-mail [email protected].

† Prince Henry’s Institute of Medical Research.‡ Murdoch Children’s Research Institute.§ Department of Surgery, Royal Children’s Hospital.| Departments of Gastroenterology and Clinical Nutrition, Royal Children’s

Hospital.⊥ Department of Paediatrics, University of Melbourne.# These authors contributed equally to the manuscript.

10.1021/pr900976f 2010 American Chemical Society Journal of Proteome Research 2010, 9, 1437–1449 1437Published on Web 11/30/2009

deepening of the crypts and thickening of the mucosa.10,11,23-26

This is accompanied by alterations in the rate of apoptosis andan increase in cellular DNA, RNA and protein content.3,10,20

Concomitant changes in nutrient uptake, ion transport anddigestive capacity ideally lead to increased absorption ofcarbohydrate, protein, water and electrolytes per unit lengthin the small intestine,3,11,25,26 although absorption per cell isdecreased due to a lack of mature cells in the villi.10 Thesechanges are most prominent in the ileum and are directlyproportional to the amount of bowel remaining.3,10 The successof the adaptive response directly determines whether a patientwill regain enteral autonomy or progress to a state of intestinalfailure. In those patients where IF is irreversible and long-termdependence on PN is considered life-threatening, intestinaltransplantation is indicated as the most appropriate -treatment.2,14-18 The key to successful transplantation lies withearly referral, prior to the development of life-threateningcomplications and the requirement for simultaneous multio-rgan transplant.2,17-19 It is therefore imperative that patientswho will not achieve enteral autonomy are distinguished assoon as possible, to facilitate the best long-term managementand patient prognosis.3,11 However, to date there is no clinicallyuseful marker with sufficient accuracy to predict at an earlystage whether patients will develop IF.

A number of factors are known to influence the success ofintestinal adaptation and have been extensively reviewed; theseinclude the composition of luminal nutrients, pancreatico-biliary secretions, secreted hormones and requirement forpeptide growth factors.2,3,8,10,13,27-30 Several studies have at-tempted to characterize gene expression following MSBR toanalyze mechanisms of intestinal adaptation,11,20,21,31 or tocharacterize the developmental progression of proliferatingcells migrating along the crypt-villus axis.32 However themechanisms underlying the successful intestinal adaptationresponse, including the molecular signals that initiate, maintainand ultimately terminate the adaptation process, remain poorlyunderstood.20,21,31 A marker of intestinal adaptation would beof significant clinical use, aiding in the evaluation of patientresponse to therapeutic interventions that may promote in-testinal adaptation as well as allowing for earlier referral ofpatients for transplantation who will not achieve ultimateenteral autonomy.

We have applied a proteomic strategy to analyze proteinexpression in a well-established porcine model of intestinaladaptation33 following MSBR. This is the first study describingthe application of proteomic technology to investigate thechanges in protein expression that occur during adaptation ofthe intestinal lumen as a result of SBS.

Experimental Section

Animals. All experiments were conducted according to theguidelines of the National Health and Medical ResearchCouncil, and with prior ethics approval from the Animal EthicsCommittee of the Murdoch Children’s Research Institute(Melbourne, Australia). Weaned 3-week old Landrace/LargeWhite Cross piglets (Victorian Institute of Agricultural Science,Werribee, Australia) were acclimatized at the Royal Children’sHospital Animal Research Laboratory (Melbourne, Australia)at a constant temperature of 22 °C with a 12 h light cycle forone week prior to surgery. Surgical procedures have beendescribed previously.33-36 In brief, 4-week old piglets under-went either a 75% proximal small bowel resection (MSBR),transection and reanastomosis (SHAM), or no operation (NOC).

Small bowel resection included removal of the small bowelfrom 1 m distal to the ligament of Treitz to 2.5 m proximal tothe ileocecal valve. The SHAM operation group were transectedand reanastomosed 2.5 m proximal to the ileocecal valve.

Following surgery all animals (both control and treatmentgroups) received identical peri- and postoperative care. Animalswere maintained in individual cages to accurately measure dietconsumption. Weight was measured weekly prior to the morn-ing feed. Serum required for fasting bile acid, cholesterol andtriglyceride measurements was collected 24 h prior to operationand at 2, 4, and 6 weeks postoperatively. Samples wereevaluated in the Department of Laboratory Medicine (RoyalChildren’s Hospital, Parkville, Australia) using establishedmethodology. After sacrifice ileal tissue was harvested, andeither placed in 10% neutral buffered formalin (AustralianBiostain Pty. Ltd., Australia) for paraffin embedding or im-mediately snap frozen. Prior to protein extraction the ilealmucosa was manually separated from the underlying muscle.

Expression Analysis and Protein Identification. Proteinextraction from snap-frozen ileal tissue, fluorescent proteinlabeling using CyDyes and two-dimensional polyacrylamide gelelectrophoresis (2D DIGE) were as described.37 Labeling wasperformed using 50 µg protein as recommended by themanufacturer. Isoelectric focusing was carried out using bothpH 3-10 and pH 5-8 gradients according to the followingparameters; 60 µA per strip, 100 V/90 min, 300 V/90 min, 500V/3 h, gradient to 1000 V/4 h, gradient to 8000 V/3 h, constant8000 V until reaching 60 000 Vh. Differential expression analysisbased on normalized spot volumes was carried out using PG240Same Spots software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK). All proteomic analysis was carried out on individualanimal samples in each group. Reciprocal labeling experimentswere also carried out using a pooled protein sample, and anyproteins showing differential labeling effects specific to the useof the Cy3/Cy5 dyes were eliminated from the analysis.

Protein spots of interest were excised using a ProPicII roboticspot picker (Genomic Solutions, MI) based on the X-Ycoordinates exported directly from PG240 SameSpots. Proteinidentification by MALDI-TOF MS and MS/MS was also asdescribed.37 Monoisotopic peak masses were automaticallyextracted using GPS Explorer software (v 3.0 build 311; AppliedBiosystems, CA) and peak lists searched against the nonredun-dant UniProtKB/Swiss-Prot database (release 57.3; 468851sequence entries; http://www.uniprot.org) using the MASCOTsearch engine (updated 03-01-2007; http://www.matrix-science.com). Species was restricted to mammalian, carbony-lamide-cysteine (CAM - fixed modification) and oxidation ofmethionine (variable modification) were taken into account, aparent ion mass tolerance of 0.1 Da and 1 missed cleavage(enzyme specificity trypsin) was allowed. Up to fifteen of themost intense peptides detected in each MS scan were auto-matically selected for MS/MS analysis. Peak lists were extractedusing Data Analysis software version 3.4 (Bruker Diagnostics,Germany). The parameters used to create the peak lists wereas follows: mass range 100 to 3000 Da; signal-to-noise thresholdof 5; minimum compounds length of 10 spectra. Combinedpeptide mass (MS) and fragmentation (MS/MS) data weresearched using in-house MASCOT search engine (version 1.1,Matrix Science) against the UniProtKB/Swiss-Prot database asabove, with fragment mass tolerance of 0.1 Da. The followingcriteria were used to evaluate the search; MOWSE score (e44for PMF data; g 72 for combined MS/MS data), number ofpeptides matched (g6 for PMF data; g2 for combined MS/MS

research articles Stephens et al.

1438 Journal of Proteome Research • Vol. 9, No. 3, 2010

data); and the estimated molecular mass and pI as determinedfrom the 2D gel.

Statistical Analysis. All statistical analyses of proteomicexpression data were performed automatically by the PG240SameSpots software. Expression data are given as meannormalized spot volume ( standard deviation. Hierarchicalclustering and protein expression heat maps were generatedusing open source R software (http://www.r-project.org/).Statistical analysis of immunoblotting and immunohistochem-istry results was carried out using SigmaPlot software (SystateSoftware Inc., Chicago, IL) with data expressed as mean (standard error of the mean (SEM).

Immunoblotting. Total protein was extracted from snap-frozen, unfixed ileal tissue using TRIzol reagent (Invitrogen, CA)according to the manufacturer’s instructions. Protein pelletswere resuspended in 0.1% SDS (w/v) and protein contentdetermined using the bicinchononic acid (BCA) protein assaykit (Pierce Biotechnology, IL). A 10 µg aliquot of protein fromeach specimen was separated in a 10% SDS PAGE tris-glycinegel, and transferred to nitrocellulose membrane (GE, Uppsala,Sweden). Proteins were detected using polyclonal antibodiesdirected against FABP-6 (1:2000; R&D Systems, Minneapolis,MN), I-FABP (1:3000; R&D Systems, Minneapolis, MN) andL-FABP (1:10 000; Abcam, Cambridge UK) or �-actin (SigmaAldrich, USA) and a secondary HRP-conjugated antibodydirected against either goat or rabbit as required. ECL reagent(GE Healthcare) was used to visualize protein detection, andimages were analyzed using ImageJ software. All quantitationwas performed relative to a �-actin control.

Immunohistochemical Staining. Immunohistochemical stain-ing was carried out on paraffin-embedded, formalin-fixedsections of ileal tissue. Monoclonal antibodies directed againstFABP-6 (1:50; R&D Systems, Minneapolis, MN), I-FABP (1:100;R&D Systems, Minneapolis, MN) and L-FABP (1:100; Abcam,Cambridge UK) were used to visualize protein localization.Sections were photographed and the number of positivelystained cells in either the crypt or villus quantitated usingImageJ software (v1.34s; http://rsb.info.nih/gov/ij). All imageanalysis was performed by an experienced histologist.

Biological Enrichment and Pathway Analysis. Proteinsidentified as differentially expressed following MSBR, relativeto both SHAM and NOC controls, were further analyzed forenrichment of specific biological processes and pathways usingGeneGO pathway analysis software (Version 4.3, Build 9311;http://www.GeneGO.com). For each identified protein, theappropriate human homologue was first located in the Uni-ProtKB/Swiss-Prot database using the online Protein IdentifierCross-Reference Service (http://www.ebi.ac.uk/Tools/picr/search.do).38 The accession numbers of these human homo-

logues were then uploaded into the GeneGO environmentalong with specific proteomic fold change data. Pathwayanalysis and GO ontologies were determined using the manu-ally curated MetaCore database. Ranking of relevant pathwaysand GeneGO processes was based on hypergeometric distribu-tion p values as performed by the software, with a p value ofe10-6 considered significant.

Results

Recovery from Surgery and Characterization of theAdaptive Response in MSBR Piglets. To evaluate the recoveryof animals following surgery, the nutrient intake and weightgain of piglets from the NOC, SHAM, and MSBR groups weremonitored over a 6 week recovery period. During the initial 2weeks following surgery, piglets in the MSBR group had areduced energy intake compared to piglets from either of theSHAM or NOC groups; by postoperative week 4, however, theenergy intake of all animals was comparable (Figure 1a). Priorto surgery all animals were of comparable weight, with pigletsthat underwent MSBR initially showing a reduced rate of weightgain compared to NOC and SHAM animals (Figure 1b). Whilethe absolute weight of animals in the MSBR group remainedlower than that of animals in the NOC or SHAM groups, bypostoperative week 5 the rate of weight gain in each group wassimilar (Figure 1b), indicating that animals that had undergoneMSBR made a successful recovery following surgery.

Following surgical bowel resection there is a diminishedcapacity of the remaining bowel to absorb lipids and reclaimluminal bile acids.39 Therefore, the levels of serum bile acids,triglycerides, and cholesterol were assessed throughout thepostoperative period. Neither serum bile acid nor triglyceridelevels altered significantly in SHAM-operated animals com-pared to the NOC group (Figure 2a,b). By contrast, serum bileacid and triglyceride levels were significantly increased in theMSBR animals relative to both SHAM and NOC groups (Figure2a,b; serum bile acids: p < 0.01 vs NOC, p < 0.05 vs sham; serumtriglyceride: p < 0.001 vs. NOC, p < 0.01 vs sham). There wasno significant difference in serum cholesterol levels observedbetween any of the experimental groups (Figure 2c).

Next, a microscopic evaluation of the intestinal villi and cryptsurface areas was undertaken to determine whether morpho-logical changes in the intestinal mucosa, characteristic ofintestinal adaptation, had occurred in the MSBR group. Animalsin both the SHAM and NOC groups displayed no apparentdifference in the total villus or crypt area after the 6 weekrecovery period; by contrast, both crypt and villus areas wereincreased in MSBR animals (Figures 3a and b). Lengthening ofthe villi and deepening of the intestinal crypts is a well-

Figure 1. Characterization of dietary intakes in piglets following surgery. (A) Energy and (B) total weight gain were measured in pigletsover the 6-week experimental period. NOC, nonoperational controls; SHAM, sham-operated controls; MSBR, massive small bowelresection group. Mean ( SEM; n ) 6/group.

Fatty Acid Binding Protein Expression from Intestinal Adaptation research articles

Journal of Proteome Research • Vol. 9, No. 3, 2010 1439

characterized adaptive response following surgery.10,11,23-26

Taken together, the data indicate that piglets in the MSBR

group displayed intestinal adaptation following a 75% loss insmall intestine length.

Proteomic Expression Analysis Reveals Specific Changesin MSBR Animals Reflecting Intestinal Adaptation. Ileal tissuefrom the NOC, SHAM and MSBR groups was extracted and theprotein expression compared by 2D DIGE across both pH 3-10and pH 5-8 gradients (Figure 4a and b). Proteins displayingsignificant expression changes in MSBR animals (p e 0.01;ANOVA) of greater than 1.5-fold compared to either the NOCor SHAM groups were considered to be differentially expressed.The results of the two individual proteomic expression analysesare summarized in Table 1. A total of 71 and 53 significantdifferences between the three groups were observed across thepH 3-10 and 5-8 ranges, respectively (Table 1). The majorityof these represented increased protein expression levels inMSBR animals relative to both of the NOC and SHAM groups.Of these differences, 59 (pH 3-10), and 45 (pH 5-8), weresignificantly altered in MSBR animals relative to both of the

Figure 2. Bile acids, triglyceride and cholesterol levels in pigletsfollowing surgery. Total serum levels of bile acids, triglyceridesand cholesterol were measured in piglets throughout the 6 weekrecovery period following surgery. NOC, nonoperational controls;SHAM, sham-operated controls; MSBR, massive small bowelresection group. Mean ( SEM; n ) 6/group. t test; *p e 0.05;**p e 0.01, ***p e 0.001.

Figure 3. Changes in crypt and villus area in piglets followingMSBR. Total (A) crypt and (B) villus area in isolated segments ofileal tissue was measured in NOC (nonoperational controls),SHAM (sham-operated controls) and MSBR (massive small bowelresection) piglet groups. Mean ( SEM; n ) 6/group. t test; ***pe 0.001.

Figure 4. Representative 2D PAGE images showing differentiallyexpressed proteins following MSBR. Isolated proteins from NOC,SHAM and MSBR animals were analyzed by 2D DIGE in both pH3-10 (A) and pH 5-8 (B) gradients. Significantly (p e 0.01)differentially expressed proteins in MSBR animals relative to bothNOC and SHAM groups are indicated.

Table 1. Summary of Proteomic Expression Data FollowingIntestinal Adaptationa

Experiment pH 3-10Total # spots observed 1223Total differences observed 71increased in MSBR vs NOC 58decreased in MSBR vs NOC 13increased in MSBR vs SHAM 48decreased in MSBR vs SHAM 11

Biological variabilitywithin group (% CV)

NOC 26%SHAM 30%MSBR 23%

Experiment pH 5-8Total #spots observed 1533Total differences observed 53increased in MSBR vs NOC 35decreased in MSBR vs NOC 18increased in MSBR vs SHAM 30decreased in MSBR vs SHAM 15

Biological variabilitywithin group (% CV)

NOC 25%SHAM 31%MSBR 24%

a Isolated ileal tissue from animals that underwent either no operation(NOC), control intestinal resection and anastomosis (SHAM) or 75%massive small bowel resection (MSBR) were compared by 2D DIGEacross pH 3-10 and pH 5-8 gradients. Differentially expressed proteinswere deemed to be those showing significant (p e 0.01; ANOVA)expression changes of g1.5 fold between the groups. Biological variabilitybetween individuals in each group was assessed as average CV for allprotein spots resolved in each data set.



SHAM and NOC controls (Table 1). These proteins, represent-ing expression changes specific to MSBR animals, were flaggedfor subsequent analyses. Biological variation between individu-als within each group, as assessed by coefficient of variance(CV) in normalized protein spot volumes, was consistentlybetween ∼20-30% across the two experiments (Table 1)indicating good reproducibility within the model.

Unsupervised hierarchical cluster analysis was then con-ducted using log normalized protein expression levels fromeach proteomic data set. In each case, animals that underwentMSBR consistently clustered into a single discrete group, basedon protein expression, relative to both SHAM and NOC animals(Figure 5a and b). By contrast, SHAM and NOC animals werenot separated from each other suggesting that the controlsurgical procedure involving transaction and reanastomosis ofthe small intestine (SHAM) did not significantly affect proteinexpression associated with intestinal function relative to thenonoperated controls (NOC). Together, this demonstrates thatsignificantly altered protein expression in the ileum followingMSBR could clearly discriminate animals undergoing intestinaladaptation from nonresected animals.

Differentially expressed proteins were then submitted foridentification by mass spectrometry. Of a total of 107 proteinspots analyzed (including 3 additional spots that were signifi-cantly different between MSBR and NOC, but not SHAM,animals), 63 were unambiguously identified and a further 20were reported as mixtures of 2 or more proteins, representingan overall total of 62 unique proteins (Table 2). Twelve proteinswere independently identified in both pH 3-10 and pH 5-8profiling experiments, while a further 24 protein spots (16 frompH 3-10; 8 from pH 5-8) remained unidentified and were notexamined further.

Validation of Fatty Acid Binding Protein Expression andLocalization. In the context of intestinal adaptation, fatty acidmetabolism is of particular interest as the FABP family ofproteins are key modulators of intracellular nonesterified fattyacids, bile salts and the bile acid pool. In particular, these

proteins have been proposed as a potential link betweenintestinal resection, successful adaptive response and PN-associated liver disease. Three of the fatty acid binding proteinsidentified by proteomic analysissliver fatty acid binding protein(L-FABP), intestinal fatty acid binding protein (I-FABP) andgastrotropin (FABP-6)swere independently identified in bothpH 3-10 and pH 5-8 proteomic profiling experiments. Wetherefore chose to concentrate on the expression changesobserved for these proteins.

To validate the expression changes observed by 2D DIGE,immunoblotting was carried out using antibodies againstL-FABP, FABP-6 and I-FABP (Figure 6). In each case, immu-noblotting was performed on isolated ileal tissue from 6animals in each group. Increased total expression of bothL-FABP (Figure 6a; right panel) and FABP-6 (Figure 6b; rightpanel) was observed by immunoblot in MSBR animals relativeto both NOC and SHAM controls, confirming the changesobserved in proteomic expression data (Figure 6a and b; left-hand panel). By contrast, I-FABP showed no change in MSBRanimals relative to NOC (Figure 6c; right panel), contradictingthe changes observed by proteomic profiling (Figure 6c; left-hand panel). Therefore, changes in total expression of L-FABPand FABP-6, but not I-FABP, were confirmed in MSBR animalsfollowing the 6 week recovery period.

To further characterize the effects of MSBR on intestinalFABP expression, immunohistochemical staining against eachof the FABP’s was carried out on ileal tissue sections from NOC,SHAM and MSBR animals and the number of positively stainedcells within both the crypt and villus quantified (Figure 7). Sincethe crypt drives cell renewal within the intestine, changes incell numbers within the crypt reflect influences on cell growthwhereas changes in villus cell numbers reflect increasedabsorption and intestinal function. MSBR animals displayedincreased numbers of L-FABP-positive cells within both thecrypts and villi compared to SHAM and NOC control groups(Figure 7a), again confirming the increase in total L-FABPexpression. By contrast increased numbers of FABP-6-positivecells were observed in the villi of MSBR animals, but not inthe crypts (Figure 7b) compared to both SHAM and NOC. Thissuggests that either site-specific expression changes or alteredFABP-6 localization has occurred within the intestinal epithe-lium following adaptation. No significant change in either totalexpression or localization of I-FABP was observed in MSBRanimals relative to NOC controls (Figure 7c).

Biological Expression Profiling and Enrichment Analysis. Toidentify key molecular and biological functions that were over-represented within the data set, all proteins identified wereanalyzed using GeneGO biological pathway profiling software.Enrichment analysis was first performed to identify differen-tially regulated canonical pathways that were over-representedby the proteins involved in intestinal adaptation. Five well-defined canonical pathways were identified as significantlyenriched within the data set (Table 3; also see SupplementaryData S1). Of these, four specifically related to the regulation ofcytoskeletal remodelling via actin, tubulin and keratin inter-mediate filaments (pathways 1, 2, 4 and 5; Table 3) and involvedkey pathways mediating intracellular cytoskeletal rearrange-ments.40 The fifth pathway identified involved increased mi-tochondrial oxidative phosphorylation. These biological pro-cesses would be expected of adapting small intestine, whichundergoes significantly increased cell proliferation and cytosk-eletal rearrangements relative to nonadapting intestine.26

Figure 5. Unsupervised hierarchical clustering and expressionmapping of proteins observed in proteomic profiles. Log-transformed normalized protein spot volumes were used toperform unsupervised hierarchical cluster analysis. Green indi-cates decreased expression; red indicates increased expression.Operation group (NOC, SHAM or MSBR) and pH range isindicated.



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10-

4

63C

O6A

3_H

UM

AN

non

eli

sted

CO

L6A

3C

olla

gen

alp

ha-

3(V

I)ch

ain

107

154.

6%34

3.3/

6.4

1.6

2.0

9.91

×10

-4

11b

CO

F1_

HU

MA

Nn

one

list

edC

FL1

Co

filin

-126

311

53.0

%18

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.22

-2.

7-

2.3

2.71

×10

-3

11b

CO

F2_

HU

MA

NQ

5G6

V9

CF

L2C

ofi

lin-2

139

734

.3%

18.7

/7.6

6-

2.7

-2.

32.

71×

10-

3

20D

ER

M_B

OV

INP

4584

6D

PT

Der

mat

op

on

tin

136

29.

5%24

.0/4

.86

-2.

3-

2.5

2.74

×10

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7D

ER

M_P

IGP

4584

6D

PT

Der

mat

op

on

tin

963

11.5

%22

.0/4

.86

-2.

2-

2.9

6.79

×10

-4

227a

EF

1A1_

CR

IGR

non

eli

sted

EE

F1A

1E

lon

gati

on

fact

or

1-al

ph

a1

132

1018

.4%

50.1

/9.1

1.5

1.5

3.86

×10

-3

191

EF

2_B

OV

INn

one

list

edE

EF

2E

lon

gati

on

fact

or

263

923

24.2

%95

.3/6

.41

1.5

1.6

2.81

×10

-3

221

EF

2_P

ON

AB

non

eli

sted

EE

F2

Elo

nga

tio

nfa

cto

r2

169

1314

.3%

95.3

/6.4

11.

51.

52.

04×

10-

3

223

EF

2_P

ON

AB

non

eli

sted

EE

F2

Elo

nga

tio

nfa

cto

r2

267

1921

.3%

95.3

/6.4

11.

51.

58.

97×

10-

4

394

FA

BP

6_P

IGP

1028

9F

AB

P6

Gas

tro

tro

pin

580

1275

.0%

14.2

/6.8

41.

71.

28.

40×

10-

2

5F

AB

PI_

PIG

Q45

KW

7F

AB

P2

Fat

tyac

id-b

ind

ing

pro

tein

,in

test

inal

638

1160

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15.2

/6.6

23.

12.

15.

46×

10-

3

1F

AB

PL_

PIG

P49

924

FA

BP

1F

atty

acid

-bin

din

gp

rote

in,

liver

414

646

.5%

14.1

/6.5

93.

32.

42.

83×

10-

3

6F

AB

PL_

PIG

P49

924

FA

BP

1F

atty

acid

-bin

din

gp

rote

in,

liver

347

646

.5%

14.1

/6.5

92.

92.

12.

58×

10-

3

93a

FE

TU

A_P

IGP

2970

0A

HSG

Alp

ha-

2-H

S-gl

yco

pro

tein

146

37.

7%38

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.51.

81.

84.

17×

10-

4

194a

GA

NA

B_P

IGP

7940

3G

AN

AB

Neu

tral

alp

ha-

glu

cosi

das

eA

B14

615

15.4

%10

6.6/

5.64

1.6

1.5

6.31

×10

-3

177a

GD

IB_P

IGQ

6Q7J

2G

DIB

Rab

GD

Pd

isso

ciat

ion

inh

ibit

or

bet

a18

17

18.4

%50

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.31

1.6

1.5

2.01

×10

-3

92a

GR

P78

_BO

VIN

P34

935

HSP

A5

78kD

agl

uco

se-r

egu

late

dp

rote

in31

917

27.6

%72

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.07

1.8

1.5

7.60

×10

-3

120

GR

P78

_BO

VIN

P34

935

(fra

gmen

t)H

SPA

578

kDa

glu

cose

-reg

ula

ted

pro

tein

492

2032

.4%

72.4

/5.0

71.

71.

61.

70×

10-

3

113

GR

P78

_HU

MA

NP

3493

5H

SPA

578

kDa

glu

cose

-reg

ula

ted

pro

tein

1280

3454

.6%

72.3

/5.0

71.

71.

64.

79×

10-

3

128

GR

P78

_HU

MA

NP

3493

5H

SPA

578

kDa

glu

cose

regu

late

dp

rote

in17

119

42.5

%72

.4/5

.07

1.1

1.7

9.18

×10

-3

174

GR

P78

_HU

MA

NP

3493

5H

SPA

578

kDa

glu

cose

-reg

ula

ted

pro

tein

1380

3150

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72.3

/5.0

71.

61.

51.

41×

10-

3

124

HS9

0A_B

OV

INn

one

list

edH

SP90

AA

1H

eat

sho

ckp

rote

inH

SP90

-alp

ha

214

1420

.7%

84.7

/4.9

31.

71.

73.

47×

10-

4

239a

HSP

B1_

PIG

Q5S

1U1

HSP

B1

Hea

tsh

ock

pro

tein

bet

a-1

235

840

.6%

22.9

/6.2

3-

1.5

-1.

53.

43×

10-

3

84a

K1C

20_P

IGQ

2921

8K

RT

20K

erat

in,

typ

eI

cyto

skel

etal

2022

48

57.0

%12

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.81.

91.

65.

70×

10-

6

177a

K1C

20_P

IGQ

2921

8K

RT

20K

erat

in,

typ

eI

cyto

skel

etal

2021

89

74.8

%12

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.81.

61.

52.

01×

10-

3

192a

K1C

20_P

IGQ

2921

8K

RT

20K

erat

in,

typ

eI

cyto

skel

etal

2010

95

35.5

%12

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61.

52.

16×

10-

3

239a

KC

Y_P

IGQ

2956

1C

MP

K1

UM

P-C

MP

kin

ase

747

39.3

%22

.2/6

.02

-1.

5-

1.5

3.43

×10

-3

115

MLR

N_H

UM

AN

P29

269

MY

L9M

yosi

nre

gula

tory

ligh

tp

oly

pep

tid

e9

405

844

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19.8

/4.8

-1.

7-

1.7

1.72

×10

-3

194a

MV

P_H

UM

AN

non

eli

sted

MV

PM

ajo

rva

ult

pro

tein

155

1314

.8%

99.3

/5.6

41.

61.

56.

31×

10-

3

239a

PA

RK

7_B

OV

INQ

0R67

8P

AR

K7

Pro

tein

DJ-

133

47

40.7

%20

.0/6

.84

-1.

5-

1.5

3.43

×10

-3

43P

DIA

1_B

OV

INn

one

list

edP

4HB

Pro

tein

dis

ulfi

de-

iso

mer

ase

478

1326

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57.2

/4.8

2.1

1.9

2.19

×10

-4

93a

PD

IA1_

MA

CF

Un

one

list

edP

4HB

Pro

tein

dis

ulfi

de-

iso

mer

ase

A1

133

1020

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57.3

/4.6

91.

81.

84.

17×

10-

4

362

PD

IA1_

MO

USE

non

eli

sted

P4H

BP

rote

ind

isu

lfid

e-is

om

eras

e38

613

25.0

%57

.1/4

.79

1.7

1.8

7.32

×10

-2

207

PD

IA3_

HU

MA

Nn

one

list

edP

DIA

3P

rote

ind

isu

lfid

e-is

om

eras

eA

399

815

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56.7

/5.9

81.

51.

62.

69×

10-

4

92a

PD

IA4_

HU

MA

Nn

one

list

edP

DIA

4P

rote

ind

isu

lfid

e-is

om

eras

eA

415

315

17.7

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.96

1.8

1.5

7.60

×10

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Tab

le2.

Co

nti

nu

ed

pro

tein

spo

t#

iden

tifi

edU

niP

RO

Tac

c.(S

us

scro

fa)

gen

ep

rote

inM

OW

SEsc

ore

no

.p

epti

des

iden

tifi

edse

qu

ence

cove

rage

%m

ass/

pI

fold

chan

gevs

NO

Cfo

ldch

ange

vsSH

AM

AN

OV

A

158a

PD

IA6_

PO

NA

Bn

one

list

edP

DIA

6P

rote

ind

isu

lfid

e-is

om

eras

eA

650

28

18.9

%48

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.88

1.7

1.6

2.89

×10

-4

116a

PE

BP

1_M

AC

FA

non

eli

sted

PE

BP

1P

ho

sph

atid

ylet

han

ola

min

e-b

ind

ing

pro

tein

135

94

26.7

%20

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6-

1.7

4.77

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-3

70a

QC

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HU

MA

Nn

one

list

edU

QC

RC

1C

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chro

me

b-c1

com

ple

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bu

nit

1,m

ito

cho

nd

rial

803

4.4%

52.6

/5.9

41.

91.

65.

68×

10-

3

116a

RA

C2_

MO

USE

non

eli

sted

RA

C2

Ras

-rel

ated

C3

bo

tulin

um

toxi

nsu

bst

rate

272

314

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21.4

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2-

1.6

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74.

77×

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3

239a

RB

11A

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NF

AQ

52N

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AB

11A

Ras

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ated

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Rab

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303

836

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24.4

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2-

1.5

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53.

43×

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3

4T

AG

L_B

OV

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one

list

edT

AG

LNT

ran

sgel

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116

58.2

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.87

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1-

2.5

7.50

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-5

70a

TB

A1C

_HU

MA

Nn

one

list

edT

UB

A1C

Tu

bu

linal

ph

a-1C

chai

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412

35.9

%49

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.96

1.9

1.6

5.68

×10

-3

217a

TC

PA

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IGR

non

eli

sted

CC

T1

T-c

om

ple

xp

rote

in1

sub

un

ital

ph

a14

17

13.8

%60

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.71

1.5

1.5

4.14

×10

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217a

TC

PE

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USE

non

eli

sted

CC

T5

T-c

om

ple

xp

rote

in1

sub

un

itep

silo

n17

310

13.9

%59

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.72

1.5

1.5

4.14

×10

-4

13T

CT

P_P

IGP

6128

8T

PT

1T

ran

slat

ion

ally

con

tro

lled

tum

or

pro

tein

7310

40%

19.7

/4.8

41.

2-

2.2

<0.0

0145

TE

RA

_HU

MA

NP

0397

4V

CP

Tra

nsi

tio

nal

end

op

lasm

icre

ticu

lum

AT

Pas

e15

515

15.0

%89

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.14

-1.

8-

2.1

9.74

×10

-3

177a

TX

ND

5_H

UM

AN

non

eli

sted

TX

ND

C5

Th

iore

do

xin

do

mai

n-c

on

tain

ing

pro

tein

511

07

12.0

%47

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.63

1.6

1.5

2.01

×10

-3

19V

ILI_

PIG

Q29

261

VIL

1V

illin

-1(f

ragm

ent)

742

16.8

%92

.7/5

.99

2.5

1.9

5.81

×10

-3

Fro

mp

H5-

8P

rofi

ling

167

AN

XA

4_P

IGP

0813

2A

NX

A4

An

nex

inA

444

725

%36

.0/5

.71

1.7

1.5

2.95

×10

-3

214

AN

XA

4_P

IGP

0813

2A

NX

A4

An

nex

inA

439

416

47.6

%35

.8/5

.71

1.6

1.5

1.30

×10

-2

104

AP

OA

4_P

IGO

4640

9A

PO

A4

Ap

olip

op

rote

inA

-IV

6815

35%

43.3

/5.6

92.

01.

81.

12×

10-

3

215

AT

PB

_HU

MA

Nn

one

list

edA

TP

5BA

TP

Syn

thas

esu

bu

nit

bet

a,m

ito

cho

nd

rial

107

1230

.2%

56.3

/5.1

91.

51.

62.

09×

10-

3

229a

AT

PB

_MO

USE

non

eli

sted

AT

P5B

AT

Psy

nth

ase

sub

un

itb

eta,

mit

och

on

dri

a39

713

33.1

%56

.3/5

.19

1.6

1.5

3.00

×10

-3

186

AT

PB

_RA

Tn

one

list

edA

TP

5BA

TP

Syn

thas

esu

bu

nit

bet

a,m

ito

cho

nd

rial

146

1438

.2%

56.3

/5.1

91.

51.

79.

39×

10-

4

107

CA

LR_P

IGP

2849

1C

ALR

Cal

reti

culin

481

960

.0%

11.9

/5.6

2.0

2.0

5.96

×10

-4

135a

CA

LR_P

IGP

2849

1C

ALR

Cal

reti

culin

192

853

.3%

12.0

/5.6

1.9

1.9

5.79

×10

-4

136

DE

SM_P

IGP

0254

0D

ES

Des

min

770

2759

.0%

53.6

/5.2

11.

91.

93.

66×

10-

3

140

DE

SM_P

IGP

0254

0D

ES

Des

min

1140

3066

.0%

53.6

/5.2

11.

91.

71.

23×

10-

2

166

DE

SM_P

IGP

0254

0D

ES

Des

min

694

2450

.5%

53.6

/5.2

11.

71.

73.

66×

10-

3

219

DE

SM_P

IGP

0254

0D

ES

Des

min

608

2343

.9%

53.6

/5.2

11.

61.

61.

10×

10-

2

229a

DE

SM_P

IGP

0254

0D

ES

Des

min

737

2343

.9%

53.6

/5.2

11.

61.

53.

00×

10-

3

229a

DE

SM_P

IGP

0254

0D

ES

Des

min

110

1324

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53.6

/5.2

11.

61.

53.

00×

10-

3

264

EF

2_B

OV

INn

one

list

edE

EF

2E

lon

gati

on

fact

or

274

523

25.1

%95

.3/6

.41

1.5

1.5

1.45

×10

-2

169a

FA

BP

4_P

IGO

9778

8F

AB

P4

Fat

tyac

id-b

ind

ing

pro

tein

,ad

ipo

cyte

205

639

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14.7

/6.2

9-

1.7

-1.

71.

49×

10-

2

17F

AB

P6_

PIG

P10

289

FA

BP

6G

astr

otr

op

in42

910

53.9

%14

.2/6

.84

3.2

2.4

2.57

×10

-3

41F

AB

PH

_PIG

O02

772

FA

BP

3F

atty

acid

-bin

din

gp

rote

in,

hea

rt43

17

42.1

%14

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.11

-2.

6-

2.5

3.12

×10

-4

169a

FA

BP

I_P

IGQ

45K

W7

FA

BP

2F

atty

acid

-bin

din

gp

rote

in,

inte

stin

al15

76

40.2

%15

.2/6

.62

-1.

7-

1.7

1.49

×10

-2

53F

AB

PL_

PIG

P49

924

FA

BP

1F

atty

acid

-bin

din

gp

rote

in,

liver

474

863

.8%

14.1

/6.5

92.

42.

02.

18×

10-

3

257

GR

P78

_BO

VIN

P34

935

HSP

A5

78kD

agl

uco

sere

gula

ted

pro

tein

7212

22.9

%72

.4/5

.07

1.5

1.5

1.74

×10

-3

261

GR

P78

_BO

VIN

P34

935

HSP

A5

78kD

agl

uco

sere

gula

ted

pro

tein

114

1428

.1%

72.4

/5.0

71.

51.

59.

04×

10-

3

238

GR

P78

_HU

MA

NP

3493

5H

SPA

578

kDa

glu

cose

regu

late

dp

rote

in68

1126

.9%

72.4

/5.0

71.

51.

52.

00×

10-

3

249

GR

P78

_HU

MA

NP

3493

5H

SPA

578

kDa

glu

cose

-reg

ula

ted

pro

tein

1460

3453

.8%

72.3

/5.0

71.

51.

58.

69×

10-

3

21H

2B1B

_MO

USE

non

eli

sted

His

t1h

2bb

His

ton

eH

2Bty

pe

1-B

278

958

.7%

13.9

/10.

313.

01.

52.

83×

10-

4

20H

4_H

UM

AN

P62

802

H4

His

ton

eH

490

438

.8%

11.4

/11.

363.

02.

13.

51×

10-

3

122a

H4_

HU

MA

NP

6280

2H

4H

isto

ne

H4

180

438

.8%

11.4

/11.

36-

1.9

-2.

09.

97×

10-

3

381

HB

B_P

IGP

0206

7H

BB

Hem

ogl

ob

insu

bu

nit

bet

a82

69

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Identified proteins were then used as core molecules togenerate biological networks, based on known molecularinteractions in the Metacore database. The five most signifi-cantly ranked networks are shown in Table 4 (also seeSupplementary Data S2). The two top-ranked networks identi-fied were highly enriched for processes involved in the regula-tion of lipid transport and metabolism,41 while the remainingthree were enriched for general processes of cell proliferationand growth and included central foci around p53 (Network 3;Supplementary Data S2) and the focal adhesion kinase FAK(network 5; Supplementary Data S2). These relationshipspresumably reflect the increased cell proliferation that char-acterizes the intestinal adaptation response.26 Taken together,the biological pathways identified suggest an important linkbetween fatty acid metabolism and regulation of cell growthin the adapting intestinal epithelium.

Discussion

In this study, we report the use of a proteomic approach toinvestigate the intestinal adaptation response in small intestinethat follows massive small bowel resection. Over 60 proteinswere identified by 2D DIGE profiling of ileal tissue from aporcine model, providing a comprehensive map of proteinexpression changes for subsequent studies of intestinal adapta-tion. Of particular interest were the observed expressionchanges in the fatty acid binding proteins, L-FABP, FABP-6 andI-FABP. These proteins are known to play important roles inthe modulation of nonesterified fatty acid concentration;indeed, we observed a significant increase in triglyceride andbile acid levels observed following MSBR, and also identifiedlipid metabolism via pathway analysis as significantly over-represented by proteins within our data set. We subsequentlychose to concentrate on the three identified fatty acid bindingproteins (L-FABP, FABP6 and I-FABP) in the context of intes-tinal adaptation.

L-FABP expression increased in MSBR animals postsurgery,and was paralleled by an increase in villus surface area andthe number of L-FABP positive cells within the crypts and villi.As the cells within the crypt are known to drive villus renewalthis may be suggestive of a coordinated relationship betweenL-FABP expression and villus expansion. L-FABP expression haspreviously been shown to be associated with the enterocyteslocated along the duodenal-to-ileal axis of the gut.42 Prolifera-tion leading to expansion of the villi surface area is a wellcharacterized adaptive response; therefore, L-FABP may provea useful marker of increasing villus surface area duringadaptation.

Total FABP-6 protein expression was also increased post-surgery in MSBR animals, concurrent with an increase in thenumber of positively stained cells in the villi. Interestingly, anearly increase in FABP-6 protein levels at 2 weeks postsurgery(see Supplementary Data Figure SF1) occurred concomitantwith increased crypt area, while immunohistochemistry studiesrevealed a significant increase in the number of FABP-6 stainedcells within the villus by week 6. The significance of thisdiscovery may be 2-fold. In the first instance, FABP-6 has beenshown in other model systems to act as a mediator of cellgrowth with the induction of FABP-6 within the crypts of infantmice followed by expression of FABP-6 within the villus.43

FABP-6 may therefore be involved in modulation of themorphological changes commonly associated with the adapta-tion response, such as increased villus height. Second theconcurrent increase in FABP-6 protein expression and serumT

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bile acids at week 6 is suggestive of a key role in bile acidmodulation. FABP-6 gene expression has previously beenshown to be upregulated by bile acids through the activationof nuclear farnesoid X receptor (FXR),44-46 which is alsoresponsible for the regulation of expression of bile acidsynthesis, transport and absorption.47 An increase in FABP-6and it is regulator FXR following small bowel resection maywork to limit intracellular bile acid overload and toxicity byacting as a bile acid sensor to activate export and absorptionmechanisms. Given the early detection of FABP-6 expressionand the potential of FABP-6 to modulate bile acid reabsorbtion,FABP-6 represents a novel, potential clinical marker or targetfor therapeutic intervention in MSBR patients which deservesfurther evaluation.

The expression changes observed by 2D DIGE profiling forI-FABP could not be independently validated; indeed, weobserved I-FABP protein spots at both increased and decreasedlevels relative to NOC and SHAM controls, and at slightly

different molecular masses (compare spot 5 in Figure 4a withspot 169 in Figure 4b). Both I-FABP and FABP4 were identifiedin spot 169; the differential expression observed by DIGE couldtherefore reflect a change in the expression of FABP4 ratherthan I-FABP. However, since I-FABP was also identified inde-pendently in the corresponding pH 3-10 experiment (whereasFABP4 was not), we chose to pursue validation of I-FABPexpression. Interestingly, Chang et al.48 also observed conflict-ing expression changes in I-FABP between cells of the cryptand villus. This suggests that specific forms of I-FABP may bepresent in different biological contexts. We also identifiedincreased mitochondrial oxidative respiration using GeneGObiological profiling, and identified a number of proteinsinvolved in mitochondrial function with altered expression inMSBR animals; increased expression of I-FABP is known topromote increased mitochondrial beta-oxidation and choles-terol uptake at a transcriptional and translational level.49 Site-dependent functional differences have previously been ob-

Figure 6. Total protein expression analysis for L-FABP, FABP-6 and I-FABP. Total protein expression changes observed for the fattyacid binding proteins observed in proteomics experiments (left-hand panels) were validated by immunoblot (right-hand panels) usingantibodies against L-FABP (A), FABP-6 (B) and I-FABP (C). Mean ( SD, n ) 6 per group. t test; *p e 0.05; **p e 0.01.



served for I-FABP;50 it remains to be seen whether I-FABPisoforms are important in the process of intestinal adaptation.

Previous gene expression profiling performed in rodentmodels has identified a limited number of differentially ex-pressed genes following intestinal resection.11,20,21,31 Recentwork by Chang et al. examined proteomic expression changesbetween cells in the intestinal crypts or villi of mice using 2DDIGE,48 as a measure of cellular development along the crypt-villus axis. In total 46 proteins were identified that were

differentially expressed between the two cell types,48 providinga global snapshot of protein expression changes associated withintestinal cellular maturation. Proteins including keratin 20,villin-1, actin, retinol binding protein II, ATP synthase subunitd, L-FABP and annexin A4 were observed to be increased inthe crypt cells relative to the villi; we also observed increasedexpression of all of these proteins in association with intestinaladaptation. Similarly, proteins including HSP90 alpha and betaand galectin 2 were decreased in crypt cells and were alsoidentified in our study as decreased in response to MSBR.Transgelin and apolipoprotein AIV have also been previouslyobserved with increased expression in crypts relative to villi;32

similarly, we observed increases in expression of these twoproteins as part of the adaptive response. The common proteinexpression changes observed between these studies and ourdata set is likely to reflect the cellular differentiation andproliferation that accompanies intestinal adaptation.

Biological pathway profiling demonstrated that our pro-teomic data set was functionally enriched for proteins involvedin lipid/fatty acid metabolism and cytoskeletal rearrangements,as has previously been observed for intestinal cells undergoingproliferation.32,48 The data suggest that increased cell prolifera-tion, coupled with increased lipid and fatty acid metabolism,is a feature of intestinal adaptation. However, an interestingfinding was the identification of steroid receptors within twoprotein networks enriched for the processes of lipid metabolism(network 1; Supplementary data S2) and regulation of apoptosis

Figure 7. Localization of fatty acid binding proteins L-FABP, FABP-6 and I-FABP by immunostaining of isolated ileal tissue. Changes inprotein localization and expression were analyzed by immunohistochemical staining of isolated ileal tissue using antibodies againstL-FABP (A), FABP-6 (B) and I-FABP (C). The number of positively stained cells in either the intestinal crypts (left hand panels) or villi(right hand panels) were quantitated. Mean +/- SD, n ) 6 per group. T-test; *p < 0.05; **p < 0.01.

Table 3. Most Significant Canonical Pathways Recognized byGeneGOa

identifiedGeneGO canonical

pathway mapnumber of

network objects significance (p)

Cytoskeleton remodeling;Regulation of actincytoskeleton by Rho GTPases

6 7.341e-09

Development; Slit-Robosignaling

6 4.162e-08

Cytoskeleton remodeling;Keratin filaments

6 1.323e-07

Oxidative phosphorylation 8 4.901e-07Cytoskeleton remodeling;

Neurofilaments5 6.090e-07

a Differentially expressed proteins were entered into the Metacoreenvironment and queried for their participation in known canonicalpathways. The number of identified proteins found within each pathwayis indicated. Pathways were scored by statistical significance and rankedaccordingly. Full pathways are provided (Supplementary Data S1).



(Network 4; Supplementary data S2) and containing commonfatty acid binding proteins. Previous evidence has suggestedthat the expression of I-FABP may be controlled in a tissue-specific and hormonally regulated manner.51 Androgen deple-tion has also been shown to inhibit intestinal adaptationfollowing MSBR, with testosterone stimulating intestinalgrowth;52 in addition, FABP-6 possesses an estrogen receptorbinding site.53 Both the androgen and estrogen-1 receptorswere predicted to be focal elements within the enriched proteinnetworks. In addition we identified the protein DJ-1 (PARK7)at decreased expression levels in MSBR animals. DJ-1 acts as aregulator of androgen receptor-dependent transcription andas a sensor of oxidative stress,54,55 suggesting an intimate linkbetween steroid metabolism, the cellular proliferation and lipidmetabolism in adapting intestine.

Conclusion

Treatment of short bowel syndrome, arising from eithercongenital defect or massive small bowel resection, continuesto present a clinical challenge in terms of patient management,long-term prognosis and economic cost. This is the first studyto apply proteomic methods to examine the intestinal adapta-tion process, necessary for patients to achieve good long-termprognosis after significant loss of intestinal surface area. Theidentification and characterization of intestinally expressedfatty acid binding proteins in the present study is promisingand provides new insight into mechanisms associated withintestinal adaptation. Whether similar expression changesassociated with these proteins can be detected in biologicalsamples such as blood, faeces or bile secretions remains to bedetermined; if successful, these may be useful in the searchfor a dynamic, clinically valuable marker of the adaptiveresponse following MSBR.

Acknowledgment. Dr. Andrew Stephens is supportedby an Ovarian Cancer Research Foundation fellowship. DrPrue Pereira-Fantini is supported on a grant provided by theMurdoch Children’s Research Institute. Professor PeterFuller and Associate Professor David Robertson are

supported by career fellowships from the National Healthand Medical Research Council of Australia.

Supporting Information Available: SupplementaryFigure SF1 showing data for changes in villus area, crypt area,and protein expression over time; supplementary data sectionsS1 and S2 showing biological pathway profiling data; supple-mentary data section S3 showing matched peptides and MSspectra for each identified protein. This material is availablefree of charge via the Internet at http://pubs.acs.org.

References(1) Buchman, A. L. Etiology and initial management of short bowel

syndrome. Gastroenterology 2006, 130 (2 Suppl 1), S5–15.(2) Duro, D.; Kamin, D.; Duggan, C. Overview of pediatric short

bowel syndrome. J. Pediatr. Gastroenterol. Nutr. 2008, 47 (Suppl1), S33–6.

(3) Pereira, P. M.; Bines, J. E. New growth factor therapies aimed atimproving intestinal adaptation in short bowel syndrome. J.Gastroenterol. Hepatol. 2006, 21 (6), 932–40.

(4) Cole, C. R.; Hansen, N. I.; Higgins, R. D.; Ziegler, T. R.; Stoll, B. J.Very low birth weight preterm infants with surgical short bowelsyndrome: incidence, morbidity and mortality, and growth out-comes at 18 to 22 months. Pediatrics 2008, 122 (3), e573-82.

(5) Goulet, O.; Baglin-Gobet, S.; Talbotec, C.; Fourcade, L.; Colomb,V.; Sauvat, F.; Jais, J. P.; Michel, J. L.; Jan, D.; Ricour, C. Outcomeand long-term growth after extensive small bowel resection in theneonatal period: a survey of 87 children. Eur. J. Pediatr. Surg. 2005,15 (2), 95–101.

(6) Goulet, O.; Ruemmele, F.; Lacaille, F.; Colomb, V. Irreversibleintestinal failure. J. Pediatr. Gastroenterol. Nutr. 2004, 38 (3), 250–69.

(7) Quiros-Tejeira, R. E.; Ament, M. E.; Reyen, L.; Herzog, F.; Merja-nian, M.; Olivares-Serrano, N.; Vargas, J. H. Long-term parenteralnutritional support and intestinal adaptation in children with shortbowel syndrome: a 25-year experience. J. Pediatr. 2004, 145 (2),157–63.

(8) Sondheimer, J. M.; Cadnapaphornchai, M.; Sontag, M.; Zerbe, G. O.Predicting the duration of dependence on parenteral nutrition afterneonatal intestinal resection. J. Pediatr. 1998, 132 (1), 80–4.

(9) Wallander, J.; Ewald, U.; Lackgren, G.; Tufveson, G.; Wahlberg, J.;Meurling, S. Extreme short bowel syndrome in neonates: anindication for small bowel transplantation. Transplant Proc. 1992,24 (3), 1230–5.

(10) Welters, C. F.; Dejong, C. H.; Deutz, N. E.; Heineman, E. Intestinaladaptation in short bowel syndrome. ANZ J. Surg. 2002, 72 (3),229–36.

Table 4. Top 5 Significant Protein Networks Predicted by GeneGOa

networkranking GO processes key network objects

totalnodes

rootnodes pathways p-value z-score

1 lipid transport (27.3%), lipoprotein particleclearance (15.9%), lipoprotein catabolicprocess (13.6%), cholesterol homeostasis(15.9%), sterol homeostasis (15.9%)

Fatty acid-binding protein,Actin cytoskeletal, DJ-1,Fetuin-A, Transgelin

50 13 0 6.56e-26 45.52

2 small GTPase mediated signal transduction(18.6%), intracellular signaling cascade(34.9%), localization (53.5%), negativeregulation of biological process (39.5%),mitotic spindle organization (7.0%)

GDI2, eEF1A1, Calnexin,Actin, ACTB

50 12 0 2.14e-23 41.56

3 T cell activation (20.0%), lymphocyte activation(22.2%), leukocyte activation (22.2%),multiorganism process (31.1%), regulation ofresponse to stimulus (24.4%)

PDIA3, Calnexin, GRP78,HSP70, Keratin 8

50 12 0 2.81e-23 41.14

4 negative regulation of apoptosis (34.6%),negative regulation of programmed cell death(34.6%), steroid metabolic process (26.9%),response to chemical stimulus (57.7%),cholesterol transport (15.4%)

Gastrotropin, HDL,L-FABP, HSP27, Keratin8/18

50 9 4 7.56e-17 32.11

5 anatomical structure formation (62.5%), cellularcomponent assembly (56.2%), cellularcomponent biogenesis (56.2%), cytoskeletonorganization (35.4%), cellular componentorganization (64.6%)

Actin cytoskeletal, DJ-1,TCP1, Arp2/3, SFRS1(SF2)

50 9 14 1.71e-16 30.78

a Differentially expressed proteins were entered into the Metacore environment and analyzed biological networks generated using the “AnalyzeNetworks” (AN) algorithm. Networks were scored by statistical significance and ranked accordingly. Full networks are provided (Supplementary Data S2).



(11) Stern, L. E.; Erwin, C. R.; Falcone, R. A.; Huang, F. S.; Kemp,C. J.; Williams, J. L.; Warner, B. W. cDNA microarray analysisof adapting bowel after intestinal resection. J. Pediatr. Surg.2001, 36 (1), 190–5.

(12) Colomb, V.; Fabeiro, M.; Dabbas, M.; Goulet, O.; Merckx, J.; Ricour,C. Central venous catheter-related infections in children on long-term home parenteral nutrition: incidence and risk factors. Clin.Nutr. 2000, 19 (5), 355–9.

(13) Andorsky, D. J.; Lund, D. P.; Lillehei, C. W.; Jaksic, T.; Dicanzio, J.;Richardson, D. S.; Collier, S. B.; Lo, C.; Duggan, C. Nutritional andother postoperative management of neonates with short bowelsyndrome correlates with clinical outcomes. J. Pediatr. 2001, 139(1), 27–33.

(14) Sudan, D. Cost and quality of life after intestinal transplantation.Gastroenterology 2006, 130 (2 Suppl 1), S158–62.

(15) Abu-Elmagd, K. M. Intestinal transplantation for short bowelsyndrome and gastrointestinal failure: current consensus, reward-ing outcomes, and practical guidelines. Gastroenterology 2006, 130(2 Suppl 1), S132–7.

(16) Ruiz, P.; Kato, T.; Tzakis, A. Current status of transplantation ofthe small intestine. Transplantation 2007, 83 (1), 1–6.

(17) Goulet, O.; Ruemmele, F. Causes and management of intestinalfailure in children. Gastroenterology 2006, 130 (2 Suppl 1), S16–28.

(18) Goulet, O.; Sauvat, F. Short bowel syndrome and intestinaltransplantation in children. Curr. Opin. Clin. Nutr. Metab. Care2006, 9 (3), 304–13.

(19) Vianna, R. M.; Mangus, R. S.; Tector, A. J. Current status of smallbowel and multivisceral transplantation. Adv. Surg. 2008, 42, 129–50.

(20) Otterburn, D. M.; Arthur, L. G.; Timmapuri, S. J.; McCahan, S. M.;Schwartz, M. Z. Proteasome gene upregulation: a possible mech-anism for intestinal adaptation. J. Pediatr. Surg. 2005, 40 (2), 377–80.

(21) Erwin, C. R.; Falcone, R. A., Jr.; Stern, L. E.; Kemp, C. J.; Warner,B. W. Analysis of intestinal adaptation gene expression by cDNAexpression arrays. JPEN J. Parenter. Enteral. Nutr. 2000, 24 (6),311–6.

(22) Marshman, E.; Booth, C.; Potten, C. S. The intestinal epithelial stemcell. Bioessays 2002, 24 (1), 91–8.

(23) Williamson, R. C. Intestinal adaptation (first of two parts). Struc-tural, functional and cytokinetic changes. N. Engl. J. Med. 1978,298 (25), 1393–402.

(24) Williamson, R. C. Intestinal adaptation (second of two parts).Mechanisms of control. N. Engl. J. Med. 1978, 298 (26), 1444–50.

(25) Falcone, R. A., Jr.; Shin, C. E.; Stern, L. E.; Wang, Z.; Erwin, C. R.;Soleimani, M.; Warner, B. W. Differential expression of ileal Na(+)/H(+) exchanger isoforms after enterectomy. J. Surg. Res. 1999, 86(2), 192–7.

(26) Helmrath, M. A.; VanderKolk, W. E.; Can, G.; Erwin, C. R.; Warner,B. W. Intestinal adaptation following massive small bowel resectionin the mouse. J. Am. Coll. Surg. 1996, 183 (5), 441–9.

(27) Kaufman, S. S.; Vanderhoof, J. A.; Young, R.; Perry, D.; Raynor,S. C.; Mack, D. R. Gastroenteric inflammation in children withulcerative colitis. Am. J. Gastroenterol. 1997, 92 (7), 1209–12.

(28) Modi, B. P.; Langer, M.; Ching, Y. A.; Valim, C.; Waterford, S. D.;Iglesias, J.; Duro, D.; Lo, C.; Jaksic, T.; Duggan, C. Improved survivalin a multidisciplinary short bowel syndrome program. J. Pediatr.Surg. 2008, 43 (1), 20–4.

(29) Haxhija, E. Q.; Yang, H.; Spencer, A. U.; Sun, X.; Teitelbaum, D. H.Intestinal epithelial cell proliferation is dependent on the site ofmassive small bowel resection. Pediatr. Surg. Int. 2007, 23 (5), 379–90.

(30) Sukhotnik, I.; Siplovich, L.; Krausz, M. M.; Shiloni, E. Peptidegrowth factors and intestinal adaptation in short bowel syndrome.Isr. Med. Assoc. J. 2003, 5 (3), 184–7.

(31) Erwin, C. R.; Jarboe, M. D.; Sartor, M. A.; Medvedovic, M.; Stringer,K. F.; Warner, B. W.; Bates, M. D. Developmental characteristicsof adapting mouse small intestine crypt cells. Gastroenterology2006, 130 (4), 1324–32.

(32) Mariadason, J. M.; Nicholas, C.; L’Italien, K. E.; Zhuang, M.; Smartt,H. J.; Heerdt, B. G.; Yang, W.; Corner, G. A.; Wilson, A. J.; Klampfer,L.; Arango, D.; Augenlicht, L. H. Gene expression profiling ofintestinal epithelial cell maturation along the crypt-villus axis.Gastroenterology 2005, 128 (4), 1081–8.

(33) Pereira-Fantini, P. M.; Nagy, E. S.; Thomas, S. L.; Taylor, R. G.;Sourial, M.; Paris, M. C.; Holst, J. J.; Fuller, P. J.; Bines, J. E. GLP-2administration results in increased proliferation but paradoxicallyan adverse outcome in a juvenile piglet model of short bowelsyndrome. J. Pediatr. Gastroenterol. Nutr. 2008, 46 (1), 20–8.

(34) Nagy, E. S.; Paris, M. C.; Taylor, R. G.; Fuller, P. J.; Sourial, M.;Justice, F.; Bines, J. E. Colostrum protein concentrate enhancesintestinal adaptation after massive small bowel resection injuvenile pigs. J. Pediatr. Gastroenterol. Nutr. 2004, 39 (5), 487–92.

(35) Paris, M. C.; Fuller, P. J.; Carstensen, B.; Nagy, E.; Taylor, R. G.;Sourial, M.; Holst, J. J.; Hartmann, B.; Binesm, J. E. Plasma GLP-2levels and intestinal markers in the juvenile pig during intestinaladaptation: effects of different diet regimens. Dig. Dis. Sci. 2004,49 (10), 1688–95.

(36) Pereira-Fantini, P. M.; Thomas, S. L.; Taylor, R. G.; Nagy, E.; Sourial,M.; Fuller, P. J.; Bines, J. E. Colostrum supplementation restoresinsulin-like growth factor-1 levels and alters muscle morphologyfollowing massive small bowel resection. JPEN J. Parenter. Enteral.Nutr. 2008, 32 (3), 266–75.

(37) Chen, J.; Hannan, N.; Mak, Y.; Nicholls, P.; Zhang, J.; Rainczuk,A.; Stanton, P.; Robertson, D.; Salamonsen, L.; Stephens, A.Proteomic Characterization of Mid-Proliferative and Mid-SecretoryHuman Endometrium. J. Proteome Res. 2009, 8, 2032–44.

(38) Cote, R. G.; Jones, P.; Martens, L.; Kerrien, S.; Reisinger, F.; Lin,Q.; Leinonen, R.; Apweiler, R.; Hermjakob, H. The Protein IdentifierCross-Referencing (PICR) service: reconciling protein identifiersacross multiple source databases. BMC Bioinformatics 2007, 8, 401.

(39) Alrefai, W. A.; Gill, R. K. Bile acid transporters: structure, function,regulation and pathophysiological implications. Pharm. Res. 2007,24 (10), 1803–23.

(40) Wong, K.; Ren, X. R.; Huang, Y. Z.; Xie, Y.; Liu, G.; Saito, H.; Tang,H.; Wen, L.; Brady-Kalnay, S. M.; Mei, L.; Wu, J. Y.; Xiong, W. C.;Rao, Y. Signal transduction in neuronal migration: roles of GTPaseactivating proteins and the small GTPase Cdc42 in the Slit-Robopathway. Cell 2001, 107 (2), 209–21.

(41) Wang, X. X.; Jiang, T.; Shen, Y.; Adorini, L.; Pruzanski, M.; Gonzalez,F. J.; Scherzer, P.; Lewis, L.; Miyazaki-Anzai, S.; Levi, M. FarnesoidX Receptor Modulates Renal Lipid Metabolism and Diet-InducedRenal Inflammation, Fibrosis, and Proteinuria. Am. J. Physiol. RenalPhysiol. 2009.

(42) Simon, T. C.; Roth, K. A.; Gordon, J. I. Use of transgenic mice tomap cis-acting elements in the liver fatty acid-binding protein gene(Fabpl) that regulate its cell lineage-specific, differentiation-dependent, and spatial patterns of expression in the gut epitheliumand in the liver acinus. J. Biol. Chem. 1993, 268 (24), 18345–58.

(43) Crossman, M. W.; Hauft, S. M.; Gordon, J. I. The mouse ileal lipid-binding protein gene: a model for studying axial patterning duringgut morphogenesis. J. Cell Biol. 1994, 126 (6), 1547–64.

(44) Fujii, H.; Nomura, M.; Kanda, T.; Amano, O.; Iseki, S.; Hatakeyama,K.; Ono, T. Cloning of a cDNA encoding rat intestinal 15 kDaprotein and its tissue distribution. Biochem. Biophys. Res. Com-mun. 1993, 190 (1), 175–80.

(45) Grober, J.; Zaghini, I.; Fujii, H.; Jones, S. A.; Kliewer, S. A.; Willson,T. M.; Ono, T.; Besnard, P. Identification of a bile acid-responsiveelement in the human ileal bile acid-binding protein gene.Involvement of the farnesoid X receptor/9-cis-retinoic acid recep-tor heterodimer. J. Biol. Chem. 1999, 274 (42), 29749–54.

(46) Kanda, T.; Foucand, L.; Nakamura, Y.; Niot, I.; Besnard, P.; Fujita,M.; Sakai, Y.; Hatakeyama, K.; Ono, T.; Fujii, H. Regulation ofexpression of human intestinal bile acid-binding protein in Caco-2cells. Biochem. J. 1998, 330 (Pt 1), 261–5.

(47) Capello, A.; Moons, L. M.; Van de Winkel, A.; Siersema, P. D.; vanDekken, H.; Kuipers, E. J.; Kusters, J. G. Bile acid-stimulatedexpression of the farnesoid X receptor enhances the immuneresponse in Barrett esophagus. Am. J. Gastroenterol. 2008, 103 (6),1510–6.

(48) Chang, J.; Chance, M. R.; Nicholas, C.; Ahmed, N.; Guilmeau, S.;Flandez, M.; Wang, D.; Byun, D. S.; Nasser, S.; Albanese, J. M.;Corner, G. A.; Heerdt, B. G.; Wilson, A. J.; Augenlicht, L. H.;Mariadason, J. M. Proteomic changes during intestinal cell matu-ration in vivo. J. Proteomics 2008, 71 (5), 530–46.

(49) Montoudis, A.; Seidman, E.; Boudreau, F.; Beaulieu, J. F.; Menard,D.; Elchebly, M.; Mailhot, G.; Sane, A. T.; Lambert, M.; Delvin, E.;Levy, E. Intestinal fatty acid binding protein regulates mitochon-drion beta-oxidation and cholesterol uptake. J. Lipid Res. 2008,49 (5), 961–72.

(50) Iiizumi, G.; Sadoya, Y.; Hino, S.; Shibuya, N.; Kawabata, H.Proteomic characterization of the site-dependent functional dif-ference in the rat small intestine. Biochim. Biophys. Acta 2007,1774 (10), 1289–98.

(51) Sato, E.; Fujii, H.; Fujita, M.; Kanda, T.; Iseki, S.; Hatakeyama, K.;Tanaka, T.; Ono, T. Tissue-specific regulation of the expression ofrat intestinal bile acid-binding protein. FEBS Lett. 1995, 374 (2),184–6.

(52) Sukhotnik, I.; Shiloni, E.; Mogilner, J.; Lurie, M.; Hirsh, M.; Coran,A. G.; Krausz, M. M. Effect of sex and sex hormones on structural



intestinal adaptation after massive small bowel resection in rats.J. Pediatr. Surg. 2005, 40 (3), 489–95.

(53) Lin, C. Y.; Vega, V. B.; Thomsen, J. S.; Zhang, T.; Kong, S. L.; Xie,M.; Chiu, K. P.; Lipovich, L.; Barnett, D. H.; Stossi, F.; Yeo, A.;George, J.; Kuznetsov, V. A.; Lee, Y. K.; Charn, T. H.; Palanisamy,N.; Miller, L. D.; Cheung, E.; Katzenellenbogen, B. S.; Ruan, Y.;Bourque, G.; Wei, C. L.; Liu, E. T. Whole-genome cartography ofestrogen receptor alpha binding sites. PLoS Genet. 2007, 3 (6), e87.

(54) Shendelman, S.; Jonason, A.; Martinat, C.; Leete, T.; Abeliovich,A. DJ-1 is a redox-dependent molecular chaperone that inhibitsalpha-synuclein aggregate formation. PLoS Biol. 2004, 2 (11), e362.

(55) Takahashi, K.; Taira, T.; Niki, T.; Seino, C.; Iguchi-Ariga, S. M.; Ariga,H. DJ-1 positively regulates the androgen receptor by impairingthe binding of PIASx alpha to the receptor. J. Biol. Chem. 2001,276 (40), 37556–63.

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Proteomic Analysis of the Intestinal Adaptation Response Reveals Altered Expression of Fatty Acid...

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