temporal proteomic analysis reveals continuous impairment of intestinal development in neonatal...

Temporal Proteomic Analysis Reveals Continuous Impairment of

Intestinal Development in Neonatal Piglets with Intrauterine Growth

Restriction

Xiaoqiu Wang,† Weizong Wu,† Gang Lin,† Defa Li,†,* Guoyao Wu,†,‡ and Junjun Wang*,†

State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100193, andDepartment of Animal Science, Texas A&M University, College Station, Texas 77843

Received September 7, 2009

Efficiency of nutrient utilization is reduced in neonates with intrauterine growth restriction (IUGR)compared with those with a normal birth weight (NBW). However, the underlying mechanisms arelargely unknown. In this study, we applied temporal proteomic approach, coupled with histologicaland biochemical analyses, to study dynamic changes of the proteome in the small intestinal mucosaof IUGR piglets during the nursing period (Days 1, 7 and 21). We identified 56 differentially expressedprotein spots between IUGR and NBW piglets. These proteins participate in key biological processes,including (1) absorption, digestion and transport of nutrients; (2) cell structure and motility; (3) glucoseand energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitaminmetabolism; (7) cellular redox homeostasis; (8) stress response; and (9) apoptosis. The results of ourtemporal proteomics analysis reveal continuous impairment of intestinal development in neonatal pigletswith IUGR. The findings have important implications for understanding metabolic defects in the smallintestine of IUGR neonates and are expected to provide new strategies to improve their survival andgrowth.

Keywords: Intrauterine growth restriction • Piglets • Intestine • Development • Temporal proteomicanalysis • Histological analysis

Introduction

Genetic, epigenetic, maternal maturity, and environmentalfactors (e.g., maternal nutrition, heat stress, disease, and toxins)are important factors affecting fetal growth and development.1

Intrauterine growth restriction (IUGR) is defined as impairedgrowth and development of the mammalian embryo/fetus orits organs during pregnancy, which can be measured as fetalor birth weight less than two standard deviation of the meanbody weight for gestational age.2 Fetal growth retardation is asignificant problem in both humans3 and livestock species.4

Of particular interest, the pig exhibits the most severe naturallyoccurring IUGR among mammalian species.4 IUGR is a majorfactor contributing to high neonatal mortality because ofimpaired development of the small intestine.5,6

The small intestine is the major organ for terminal digestionand absorption of nutrients.7 The gut is also a defense barrieragainst diet-derived pathogens, carcinogens and oxidants.8

Additionally, in mammals, the small intestine is the exclusiveorgan for endogenous synthesis of citrulline (from glutamine/glutamate and proline) and arginine,9 which has versatile roles

in growth, development and health.10 Results of our recentstudy with newborn pigs indicate that IUGR negatively affectsexpression of proteins in the small intestine of piglets at birththat are related to cellular signaling, redox balance, proteinsynthesis, and proteolysis.5 At present, little is known aboutthe effects of IUGR on the intestinal proteome during postnataldevelopment. In the swine industry, the first postnatal week isthe most critical period for neonatal survival,4 and piglets areusually weaned at 21 days of age to increase the productivityof sows.11 Therefore, the objective of this study with the pigletmodel was to quantify temporal changes in small-intestinalproteins between days 1 and 21 of life.

Materials and Methods

Piglet Model and Tissue Collection. During the entire periodof gestation, gilts (Large White sires × Landrace dams; n ) 18litters) were fed 2 kg/day of a corn and soybean meal-baseddiet and had free access to drinking water, as we describedpreviously.5 Eighteen litters of piglets (Large White × Landrace× Pietran) were spontaneously delivered from sows at term(day 114 of gestation). At birth, 1 IUGR piglet (∼ 0.7 kg) and 1normal-birth-weight (NBW; ∼ 1.3 kg) piglet were obtained fromeach of 18 litters. The selected piglets (n ) 36; 18 IUGR vs 18NBW) were positioned in the second teat pairs (known asanterior mammary glands)12 sucking milk from their ownmother for 21 days. On day 0 (D0, without sucking milk), bodyweights of all neonatal piglets were recorded immediately upon

* To whom correspondence may be addressed. Dr. Defa Li or Dr. JunjunWang: State Key Laboratory of Animal Nutrition, China Agricultural Uni-versity, No. 2. Yuanmingyuan West Road, Beijing, China, 100193. Phone: +86-10-62733588. Fax: +86-10-62733688. E-mail: [email protected] [email protected].

† China Agricultural University.‡ Texas A&M University.

924 Journal of Proteome Research 2010, 9, 924–935 10.1021/pr900747d 2010 American Chemical SocietyPublished on Web 11/26/2009

birth. On day 1 (D1), 7 (D7), and 21 (D21), neonatal piglets (6IUGR and 6 NBW piglets) from each of 6 litters were weighedand then killed by jugular puncture after anesthesia, as wedescribed previously.5 The small intestine in neonatal pigs wasdefined as the portion of the digestive tract between the pylorusand the ileocecal valve, with the first 10-cm segment beingduodenum, and the subsequent 40 and 60% of the smallintestine length below the duodenum being jejunum and ileum,respectively.5 The content of whole jejunum was rapidlyremoved with saline.13 After measuring the length and weightof the whole jejunum, approximately a 20-cm segment ofmidjejunum (the middle portion of jejunum) was obtained. A3-cm portion of jejunum was fixed in 4% formaldehyde (Sigma,St. Louis, MO) at 4 °C for histological analysis and scanningelectron microscopy (for Day 1 samples only). Mucosa fromthe remaining jejunal segment was obtained as describedpreviously,13 rapidly placed in liquid nitrogen and stored at -80°C for proteomic and Western blot analyses. The animal useprotocol was reviewed and approved by the China AgriculturalUniversity Animal Care and Use Committee.

Histology Analysis of Villus Morphology. For light micros-copy, the formaldehyde fixed samples were embedded inparaffin, sectioned and mounted on glass slides for stainingwith hematoxylin and eosin, as we described.5 Intestinalmorphology was examined with a light microscope (OlympusBX50, Japan). The villus height and width was quantified usingthe Medical Image Analysis System (MIAS) software. A mini-mum of 15 well-oriented, intact villi were measured in triplicatefor each biological sample.

For scanning electron microscopy, the formaldehyde-treatedsamples were fixed with 2.5% glutaraldehyde (Sigma, St. Louis,MO) in 0.1 M phosphate buffer (pH 7.4) for 2 h at roomtemperature. They were then washed three times for 30 minwith the same buffer, placed in 1% osmium tetroxide (Sigma,St. Louis, MO) for 1 h, and then washed again using the aboveprocedures. The fixed samples were subsequently dehydratedin a graded ethanol series (30, 50, 70, 80, 90, 95, and 100%).The samples were transferred to isoamyl acetate (Sigma, St.Louis, MO), dried in a critical point drier (HITACHI HCP-2,Japan) before being coated with gold. The specimens wereexamined by scanning electron microscopy (HITACHI S-570,Japan).

Protein Extraction of Jejunal Mucosa. Proteins were ex-tracted from the jejunum mucosa as we described.14,15 Briefly,jejunal mucosa samples were homogenized in a lysis buffer (7M Solid Urea, 2 M Thiourea, 4% CHAPS, 50 mM dithiothreitol(DTT)) containing 1% protease inhibitors (100×) (GE Health-care, Piscataway, NJ). The protease inhibitor was specificallydeveloped for sample preparation in two-dimensional electro-phoresis studies to inhibit calpain II, cathepsin B, elastase,papain, plasmin, thermolysin and trypsin. Tissues were rup-tured at 0 °C using an Ultrasonicater Model VCX 500 (Sonics& Materials, Newtown, CT) at 20% power output for 10 minwith 2-s on and 8-s off cycles. After adding 1% (v/v) nucleasemix (GE Healthcare, Piscataway, NJ), the lysed cell suspensionwas kept at room temperature for 1 h to solubilize proteins,16

followed by resonication as described above to thoroughlybreak up cell membranes. The homogenate was subsequentlycentrifuged for 10 min at 13 000g at 15 °C. The supernatantfluid was collected, and its protein concentration was deter-mined using a PlusOne 2-D Quant Kit (GE Healthcare, Piscat-away, NJ). Protein extracts were stored in aliquots (1 mg ofprotein) at -80 °C.

Two-Dimensional Gel Electrophoresis (2-DE). With one gelfor each IUGR and NBW paired sample in each of the 3 time-phases (D1, D7 and D21), a total of 18 gels were run for the2-DE using commercial IPG strips (pH 3-10 NL, 24 cm) (GEHealthcare, Piscataway, NJ) for isoelectric focusing (IEF) andthen standard vertical SDS-PAGE (12.5%) for second dimension.Briefly, mucosal extracts (1 mg protein/sample) were loadedonto IPG DryStrips using the in-gel sample rehydration tech-nique, according to the manufacturer’s instructions.16 Afterrehydration for 12 h, the first-dimensional IEF was carried outat 20 °C for 100 000 Vh in the Ettan IPGphor II IEF system (GEHealthcare, Piscataway, NJ), as we described.17 Sequentially,IPG strips were equilibrated for 15 min in 4 mL of equilibrationbuffer-1 (6 M urea, 1% DTT, 30% glycerol, and 50 mM Tris-ClpH 8.8) and then in 4 mL of equilibration buffer-2 (6 M urea,2.5% iodoacetamide, 30% glycerol, 50 mM Tris-Cl pH 8.8) for15 min. The second dimension was carried out on an EttanDALT six (GE Healthcare, Piscataway, NJ) at 30 mA/gel for 30min, and then at 50 mA/gel for about 6 h. In the second-dimensional procedure, temperature was set at 10 °C. The gelswere then stained with colloidal Coomassie Brilliant Blue G-250(Amresco, Inc., Solon, OH).

Image Analysis. High-resolution gel images (600 dpi) wereobtained using an ImageScanner Model PowerLook 2100XL(UMAX Technologies, Atlanta, GA) and image analysis wasperformed using an Image-Master 2D Platinum Version 6.01according to manufacture’s protocol (GE Healthcare, Piscat-away, NJ). After normalizing the quantity of each spot by totalvalid spot intensity, differentially expressed protein spots (P <0.05) with a deviation of over 1.5-fold in the relative volume(% vol) were selected and subjected to identification by massspectrometry (MS).

In-Gel Digestion. Protein spots of interest were manuallyobtained and destained with 100 µL of 50% (v/v) acetonitrile(ACN) in 25 mM ammonium bicarbonate for 1 h. After theprotein samples were completely dried by vacuum centrifuga-tion (Eppendorf Concentrator 5301, Germany) for 30 min, theywere digested with 2 µL of trypsin (Amresco, Inc., Solon, OH)in 25 mM ammonium bicarbonate at 4 °C for 1 h, and thenincubated at 37 °C for 12 h. The resulting peptides weresubjected to sequential extraction (3 times at 37 °C) with 8 µLeach of 5% trifluoroacetic acid (TFA) for 1 h, 2.5% TFA in 50%ACN for 1 h, and 100% ACN for 1 h. Extracted protein sampleswere dried in a vacuum centrifugation.

Protein Identification by MS and Database Search. Peptidesfrom in-gel digested proteins were mixed with a matrix solution[R-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA, and50% ACN]. MALDI-TOF/TOF MS (Matrix Assisted Laser De-sorption Ionization-Time Of Flight/Time Of Flight MS) forprotein identification was carried out on a 4700 MALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems, Foster City, CA)with 355-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) laser and 20-kV accelerated voltage. After MS, 20 parentmass peaks with 700-3200 Da of mass range and minimumsignal/noise values were selected for MS/MS analysis. Proteinidentification was achieved through Peptide Mass Fingerprint(PMF) and MS/MS data searches using GPS Explorer Worksta-tion (Applied Biosystems, Foster City, CA) with the in-housesearching engine Mascot and the searching taxonomy ofMammalia against the NCBInr database. Search parametersincluded: (1) trypsin, as the enzyme of protein digestion; (2)monoisotopic, as mass value; (3) unrestricted, as peptide mass;(4) (0.3 Da, as peptide mass tolerance; (5) oxidation (M) and

Impairment of Intestinal Development in Neonatal Piglets research articles

Journal of Proteome Research • Vol. 9, No. 2, 2010 925

carbamidomethyl (C), as variable modifications; and (6) 1, asmaximum missed cleavages. In this procedure, a protein matchwith a score >71 was considered significant (P < 0.05).

Western Blotting for Protein Analysis. Extracted proteins(30 µg/sample) were separated by electrophoresis (Bio-Rad,Richmond, CA) on 12.5% SDS-PAGE before being transferredelectrophoretically to a PVDF membrane (Millipore, Billerica,MA). After blocking with TBST (0.05% Tween 20, 100 mM Tris-HCl and 150 mM NaCl, pH 7.5) containing 5% fat-free dry milkat 4 °C overnight, the membranes were incubated with primaryantibodies, that is, anti-ALB, anti-APOA1, anti-GRP94 and anti-PRDX1 (Beijing Biosynthesis Biotechnology Co., Ltd., China)in dilution of 1:300, 1:300, 1:500 and 1:500, respectively for 2 h.The membranes were then rinsed in TBST and incubated witha secondary antibody (horseradish peroxidase-labeled anti-rabbit IgG diluted 1:1000) for 2 h. The protein bands werevisualized with a chemiluminescence subtract using a gel-imaging system (Tanon Science and Technology, Shanghai,China) with Image Analysis Software (National Institutes ofHealth, Bethesda, MD).

Statistical Analysis. Normality of the data was tested usingthe Shapiro-Wilk test in SAS (version 8.1; SAS Institute, Cary,NC). Data were analyzed by one-way analysis of variance(ANOVA) or two-way ANOVA, with each animal as an experi-mental unit. All analyses were performed using SAS. Data areexpressed as means and SEM P < 0.05 was considered statisticalsignificance.

Results

Body Weights of Piglets. Body weights of IUGR and NBWpiglets at birth (D0) were 0.73 and 1.31 kg, respectively (P <0.01; Table 1). Between D1 and 21 of life, IUGR pigletscontinued to have a lower (P < 0.01) body weight than NBWpiglets (Table 1). At D21, the body weight of IUGR piglets was27% lower (P < 0.01) than that of NBW piglets.

Jejunal Lengths and Weights. Table 2 summarizes thejejunal length and weights of IUGR and NBW piglets betweenD1 and 21 of life. The absolute jejunal length of IUGR pigletswas shorter (P < 0.01) than that of NBW piglets, at each of 3time-phases (D1, D7 and D21). Likewise, absolute jejunalweights and body weights of IUGR piglets were lower (P < 0.01)compared with NBW piglets at all time points. Compared withNBW piglets, the relative length of jejunum (jejunal length/body weight) in IUGR piglets transitioned from a higher (P <0.01) value on D0 to no difference (P > 0.05) on D7 and to alower (P < 0.01) value on D21. However, the relative weight ofjejunum was consistently lower (P < 0.01) in IUGR than in NBWpiglets at all time points.

Jejunal Villus Morphology. At D1, the villus height and widthof jejunum were lower (P < 0.05) in IUGR than in NBW piglets(Table 3). The difference in jejunal villus height between IUGRand NBW piglets was even more pronounced (P < 0.01) at D21

than at D1. Jejunal villus width did not differ (P > 0.05) betweenIUGR and NBW piglets at D7 or D21.

Representative morphologies of the jejunum from IUGR andNBW piglets are illustrated in Figure 1. At D1, scanning electronand light microscopic observations revealed thinner andatrophic villus tips in the jejunum of IUGR piglets (Figure 1,A1 and B1) compared with NBW (Figure 1, A2 and B2).Additionally, columnar epithelial cells and microvilli on thesurface of jejunal villus in IUGR piglets were sparse, hadanomalously loose array, and exhibited histological lesions(Figure 1, A1 and B1). In contrast, the jejunum of NBW pigletshad more tight cell junctions and a normally oriented mor-phology (Figure 1, A2 and B2). Light microscopic examinationshowed the presence of edema beneath the layer of columnarepithelial cells in D7 IUGR jejunum (Figure 1, C1 and C2) andabnormally obscure appearance of jejunal brush border (mi-crovilli) in D21 IUGR jejunum (Figure 1, D1 and D2).

Temporal Analysis of Proteins. A total of 56 protein spotswere differentially expressed in jejunum between IUGR andNBW piglets at D1, D7 and D21. Biochemical information aboutthese protein spots is summarized in Tables 4 and 5, whereastheir appearance on the gel images is labeled in Figure 2 andFigure 3. On the basis of their biological functions, theseproteins are classified in nine groups: (1) absorption, digestionand transport; (2) cell structure and motility; (3) glucose andenergy metabolism; (4) lipid metabolism; (5) amino acidmetabolism; (6) mineral and vitamin metabolism; (7) cell redoxhomeostasis; (8) stress response; and (9) cellular apoptosis.

Absorption, Digestion and Transport. Seven spots of pro-teins were related to absorption, digestion and transport of

Table 1. Body Weights of IUGR and NBW Piglets at Birth andduring the Suckling Perioda

body weight (kg)

piglet

body weightat birth (n ) 18per group) (kg) day 1 day 7 day 21

IUGR 0.73 ( 0.01**b 0.88 ( 0.02** 1.94 ( 0.04** 5.13 ( 0.16**NBW 1.31 ( 0.02 1.46 ( 0.05 2.64 ( 0.06 6.52 ( 0.19

a Values are means ( SEM, n ) 6 per group except for body weight atbirth. b **, P < 0.01 vs the NBW group.

Table 2. Jejunal Length and Weights of IUGR and NBWPiglets during the Suckling Perioda

pigletjejunal

length (cm)jejunal

weight (g)

jejunallength index

(cm/kg)b

jejunalweight

index (%)c

Day 1IUGR 186.7 ( 5.3**d 15.1 ( 0.33** 212.5 ( 11.2** 1.72 ( 0.03**NBW 272.5 ( 4.5 37.4 ( 0.87 187.0 ( 5.9 2.57 ( 0.12

Day 7IUGR 236.2 ( 16.3** 48.3 ( 1.4** 121.8 ( 2.0 2.49 ( 0.07**NBW 346.2 ( 8.0 80.4 ( 2.1 131.1 ( 4.0 3.06 ( 0.09

Day 21IUGR 395.5 ( 21.2** 92.1 ( 4.4** 77.1 ( 2.9** 1.79 ( 0.04**NBW 594.2 ( 12.8 152.9 ( 3.2 91.1 ( 5.6 2.35 ( 0.04

a Values are means ( SEM, n ) 6 per group. b Jejunal length index )Jejunal length/Body weight. c Jejunal weight index ) Jejunal weight/Bodyweight × 100%. d **, P < 0.01 vs the NBW group.

Table 3. Jejunal Villus Heights and Widths of IUGR and NBWPiglets during the Suckling Perioda

piglet villus height (µm) villus width (µm)

Day 1IUGR 329.8 ( 14.7**b 26.4 ( 2.0*NBW 415.7 ( 17.2 36.5 ( 0.5

Day 7IUGR 353.7 ( 16.1** 40.2 ( 1.7NBW 476.3 ( 9.3 37.1 ( 1.7

Day 21IUGR 215.3 ( 10.6** 40.3 ( 1.8NBW 386.3 ( 15.8 41.2 ( 2.1

a Values are means ( SEM, n ) 6 per group. b *, P < 0.05; **, P < 0.01vs the NBW group.

research articles Wang et al.

926 Journal of Proteome Research • Vol. 9, No. 2, 2010

nutrients. They are albumin (ALB, Spot L041, L161, L162, K081),chymodenin (Spot L2101), chymotrypsinogen B (CTRB, SpotL113), and clathrin light polypeptide isoform A (CLTA, SpotP020). Abundance of these proteins was consistently andcontinuously lower (P < 0.05) in the jejunal mucosa of IUGRpiglets compared with NBW piglets at D1, D7 an D21.

Cell Structure and Motility. Five spots of proteins playimportant roles in cell structure and motility. In comparisonwith NBW piglets, levels of ezrin (EZR, Spot K074), gamma-actin (ACTG1, Spot L1368), and keratin 8 (KRT8, Spot K082)were lower (P < 0.05) in the jejunum of IUGR piglets at bothD1 and D21, but higher (P < 0.05) at D7. In addition, COFILINprotein (CFL1, Spot K054) was down-regulated in the IUGRgroup at D1, D7 and D21. In contrast, abundance of Villin 1(VIL1, Spot K073) was down-regulated in IUGR piglets at D7.

Glucose and Energy Metabolism. Differentially expressedproteins that participate in energy metabolism include: enolase1 (ENO1, Spot K084), fructose-bisphosphate aldolase A (FBPA,Spot K087), triosephosphate isomerase (TPI1, Spot L074),mitochondrial succinate dehydrogenase complex subunit A

(SDHA, Spot L061), cytosolic glycerol-3-phosphate dehydro-genase (cGPDH, Spots L099, L1415), glycerol-3-phosphatedehydrogenase 1-like protein (GPD1L, Spot L075), cytochromeb5 (CYB5, Spot P027), cytochrome c oxidase subunit 5B,mitochondrial (COX5B, Spot P111), and creatine kinase (CK,Spots L1862, K099, K100). At D1, all of these proteins werereduced (>1.5-fold; P < 0.05) in the IUGR group. Among them,ENO1, FBPA, TPI1, cGPDH, GPD1L, CYB5, COX5B and CK werecontinuously down-regulated (P < 0.05) at D7 and/or D21,whereas cGPDH, GPD1L, CYB5, COX5B were up-regulated atD7. In addition, levels of SDHA were higher (P < 0.05) in theIUGR jejunum at both D7 and D21.

Lipid Metabolism. Five spots of proteins are related to lipidmetabolism. Abundance of apolipoprotein A-I (APOA1, SpotL094, P013) and apolipoprotein A-IV (APOA4, Spot L1146) waslower (P < 0.05) in the jejunal mucosa of IUGR piglets at D1,D7 and D21, when compared with NBW piglets. Additionally,at all 3 time-phases, levels of fatty acid binding protein 5(FABP5, Spot P121) and fatty acid-binding protein 1 (FABP1,Spot L049) were lower (P < 0.05) at D1 and D21, but nodifference was detected on D7, in comparison with NBWpiglets.

Amino Acid Metabolism. Four spots of jejunal proteinsrelated to amino acid metabolism were affected by IUGR.Compared with NBW piglets, abundance of 3-hydroxyanthra-nilate 3,4-dioxygenase (3HAO, Spot K101), aminoacylase I(ACY1, Spot K085), and S-adenosylhomocysteine hydrolase(AHCY, Spot L006) was lower (P < 0.05) in the jejunum of IUGRpiglets at D1 and D21, but was higher (P < 0.05) at D7. Incontrast, levels of mitochondrial ornithine aminotransferase(OAT, Spot K076) were consistently reduced (P < 0.05) in theIUGR jejunum at all time points.

Mineral and Vitamin Metabolism. Differentially expressedproteins related with mineral and vitamin metabolism includetransferrin (TF, Spots K038, K039, K040, K089) and retinolbinding protein 2, cellular (RBP2, Spots L137, L138). Levels ofthese proteins were lower (P < 0.05) in the jejunal mucosa ofIUGR piglets than those in NBW piglets between D1 and D21.Abundance of another related protein, hemopexin (HPX, SpotP152), was down-regulated (P < 0.05) in the IUGR jejunalmucosa at both D7 and D21. Compared with NBW piglets,levels of haptoglobin (HP, Spot L189) in IUGR piglets werereduced (P < 0.05) at D1 and D21, but were increased (P < 0.05)at D7.

Cell Redox Homeostasis. IUGR affected (P < 0.05) expressionof a number of proteins involved in regulation of cell redoxhomeostasis. Specifically, abundance of beta-globin (BG, SpotK049) and protein disulfide isomerase-associated 3 (PDIA3,Spot P006) was reduced (P < 0.05) in the IUGR jejunal mucosaat D1 and D21, respectively, in comparison with NBW piglets.Levels of peroxiredoxin-1 (PRDX1, Spot K088), peroxiredoxin-5(PRDX5, Spot P036) and chloride intracellular channel protein1 (CLIC1, Spot K090) were lower (P < 0.05) in IUGR than inNBW piglets at D7. Interestingly, abundance of peroxiredoxin-6(PRDX6, Spot L090) was reduced (P < 0.05) early at D1 in theIUGR jejunum compared with NBW piglets.

Stress Response. Expression of several jejunal proteins tostress response was affected by IUGR. Of particular interest,heat shock 70 kDa protein 8/heat shock cognate 71 kDa protein(HSPA8/HSC70, Spot P153, Spot L181) were up-regulated (P <0.05) in the IUGR group at D1, in comparison with NBW piglets.Abundance of 94 kDa glucose-regulated protein (GRP94, SpotK021) and glutathione S-transferase omega (GSTO, Spot P157)

Figure 1. Representative histological pictures of scanning electronmicroscopy (A1, A2), as well as hematoxylin- and eosin-stainedjejunum of IUGR (B1, C1, D1) and NBW (B2, C2, D2) piglets atDay 1 (B1 and B2), Day 7 (C1 and C2) and Day 21 (D1 and D2) oflife.



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CO

FIL

INp

rote

in[S

us

scro

fa]

CF

L1gi

|515

9213

520

215

.5(

4.1

22.9

(4.

416

.9(

1.2

31.8

(9.

058

.5(

6.8

43.8

(0.

5<0

.01

<0.0

5<0

.01

L136

8G

amm

a-ac

tin

[Hom

osa

pie

ns]

AC

TG

1gi

|450

1887

105

ND

2.6(

0.1

4.3(

0.1

3.7(

0.8

2.0(

0.2

6.0(

0.8

<0.0

5<0

.01

<0.0

1K

082

PR

ED

ICT

ED

:si

mila

rto

Ker

atin

8[S

us

scro

fa]

KR

T8

gi|2

2743

0407

701

3.6(

0.9

13.3

(0.

225

.8(

1.6

7.3(

1.0

8.9(

0.4

41.9

(0.

5<0

.05

<0.0

1<0

.01

K07

3P

RE

DIC

TE

D:

sim

ilar

toV

illin

1[S

us

scro

fa]

VIL

1gi

|194

0438

2615

6N

D1.

0(

0.2

6.3(

0.1

ND

4.8(

0.4

6.1(

0.2

<0.0

5<0

.01

<0.0

1

Glu

cose

and

En

ergy

Met

abo

lism

L186

2C

reat

ine

kin

ase

[Can

isfa

mil

iari

z]C

Kgi

|125

292

122

0.4(

0.1

ND

ND

5.7(

0.4

ND

ND

<0.0

1<0

.01

<0.0

1K

100

Cre

atin

eki

nas

e[S

us

scro

fa]

CK

gi|1

9401

8722

235

ND

ND

ND

4.2(

0.8

9.4(

0.2

ND

<0.0

1<0

.01

<0.0

1K

099

Cre

atin

eki

nas

e[S

us

scro

fa]

CK

gi|1

9401

8722

94N

DN

DN

D2.

1(

0.6

6.9(

0.3

ND

<0.0

1<0

.01

<0.0

1P

027

Cyt

och

rom

eb

5[O

ryct

olag

us

cun

icu

lus]

CY

B5

gi|3

5381

943

56.

9(

2.4

15.3

(0.

316

.2(

0.9

13.5

(0.

811

.6(

2.7

27.2

(1.

9<0

.01

<0.0

1<0

.01

P11

1C

yto

chro

me

co

xid

ase

sub

un

it5B

,m

ito

cho

nd

rial

[Su

ssc

rofa

]C

OX

5Bgi

|559

2621

718

45.

3(

0.3

20.0

(1.

432

.9(

0.8

17.6

(1.

012

.7(

0.4

58.4

(0.

7<0

.01

<0.0

1<0

.01

L141

5C

yto

solic

glyc

ero

l-3-

ph

osp

hat

ed

ehyd

roge

nas

e[S

us

scro

fa]

cGP

DH

gi|1

4971

4300

147

1.1(

0.1

5.6(

0.5

6.4(

0.1

4.2(

0.1

4.6(

0.6

8.6(

0.6

<0.0

1<0

.01

<0.0

1

L099

Cyt

oso

licgl

ycer

ol-

3-p

ho

sph

ate

deh

ydro

gen

ase

[Su

ssc

rofa

]cG

PD

Hgi

|149

7143

0018

81.

5(

0.2

8.4(

1.5

9.1(

0.6

5.7(

0.5

8.0(

0.4

9.3(

0.3

<0.0

5<0

.01

<0.0

5

K08

4E

no

lase

1[B

osta

uru

s]E

NO

1gi

|871

9650

112

01.

2(

0.3

1.7(

0.9

10.5

(1.

04.

2(

0.3

8.1(

1.3

21.6

(1.

1<0

.01

<0.0

1<0

.01

K08

7F

ruct

ose

-bis

ph

osp

hat

eal

do

lase

A[B

osta

uru

s]F

BP

Agi

|156

1204

7924

31.

8(

0.3

2.8(

0.2

ND

13.4

(0.

68.

8(

0.5

ND

<0.0

1<0

.01

<0.0

1

L075

Gly

cero

l-3-

ph

osp

hat

ed

ehyd

roge

nas

e1-

like

pro

tein

(GP

D1L

pro

tein

)[B

osta

uru

s]G

PD

1Lgi

|739

9038

410

30.

9(

0.4

24.2

(4.

832

.2(

4.9

12.4

(3.

123

.7(

3.0

40.2

(3.

2<0

.05

<0.0

10.

254

L061

Mit

och

on

dri

alsu

ccin

ate

deh

ydro

gen

ase

com

ple

xsu

bu

nit

A[S

us

scro

fa]

SDH

Agi

|112

9808

1921

40.

6(

0.3

4.6(

0.4

7.6(

1.2

2.8(

0.1

2.4(

0.4

2.5(

0.3

<0.0

1<0

.01

<0.0

1

L074

Tri

ose

ph

osp

hat

eis

om

eras

e[S

us

scro

fa]

TP

I1gi

|902

0040

444

54.

3(

1.1

22.4

(1.

230

.6(

6.0

21.3

(2.

625

.3(

2.4

42.2

(4.

0<0

.01

<0.0

10.

143

Lip

idM

etab

oli

smL0

94A

po

lipo

pro

tein

A-I

[Su

ssc

rofa

]A

PO

A1

gi|1

6435

929

52.

2(

0.2

13.4

(2.

521

.2(

2.1

22.1

(8.

023

.6(

3.0

41.0

(7.

3<0

.01

<0.0

10.

527

P01

3A

po

lipo

pro

tein

A-I

[Su

ssc

rofa

]A

PO

A1

gi|1

6435

939

55.

7(

1.9

13.8

(2.

127

.1(

9.0

10.4

(1.

630

.5(

6.9

46.6

(5/

0<0

.01

<0.0

10.

346

L114

6A

po

lipo

pro

tein

A-I

V[S

us

scro

fa]

AP

OA

4gi

|475

2383

033

4N

D1.

1(

0.3

2.9(

0.3

1.8(

0.1

3.1(

0.6

6.1(

0.3

<0.0

1<0

.01

0.10

1P

121

Fat

tyac

idb

ind

ing

pro

tein

5[S

us

scro

fa]

FA

BP

5gi

|898

8616

710

72.

2(

0.6

8.1(

1.7

3.3(

0.1

6.0(

0.8

7.1(

0.7

16.5

(2.

7<0

.01

<0.0

1<0

.01

L049

Fat

tyac

id-b

ind

ing

pro

tein

[Su

ssc

rofa

]F

AB

P1

gi|5

5742

707

141

14.6

(5.

310

1.5(

31.2

234.

2(

28.5

87.8

(4.

611

2.1(

26.1

246.

6(

19.9

<0.0

5<0

.01

0.33

9

Am

ino

Aci

dM

etab

oli

smK

101

3-h

ydro

xyan

thra

nila

te3,

4-d

ioxy

gen

ase

[Bos

tau

rus]

3HA

Ogi

|115

4958

3517

64.

3(

0.7

10.1

(1.

96.

6(

1.4

8.7(

0.2

4.1(

1.1

14.1

(0.

7<0

.05

<0.0

5<0

.01



Tab

le4

Co

nti

nu

ed

exp

ress

ion

leve

l(

×10

4 )c

IUG

RN

BW

P-v

alu

e

spo

tn

o.a

pro

tein

nam

eab

br.

acce

ssio

nn

o.

pro

tein

sco

reb

D1

D7

D21

D1

D7

D21

IUG

Rag

eIU

GR

×ag

e

K08

5A

min

oac

ylas

eI

[Su

ssc

rofa

]A

CY

1gi

|475

2269

048

42.

1(

0.3

7.5(

1.7

8.1(

1.8

10.1

(1.

22.

9(

0.4

9.5(

1.8

<0.0

5<0

.05

<0.0

1K

076

Mit

och

on

dri

alo

rnit

hin

eam

ino

tran

sfer

ase

[Su

ssc

rofa

]O

AT

gi|1

6624

4455

150

ND

0.3(

0.1

3.8(

0.7

ND

2.0(

0.5

6.1(

0.9

<0.0

1<0

.01

0.08

3

L006

S-ad

eno

sylh

om

ocy

stei

ne

hyd

rola

se[S

us

scro

fa]

AH

CY

gi|5

8801

555

135

2.1(

0.2

6.3(

1.0

5.7(

0.9

19.1

(1.

64.

8(

0.9

7.1(

0.7

<0.0

1<0

.01

<0.0

1

Min

eral

and

Vit

amin

Met

abo

lism

L189

Hap

togl

ob

in[S

us

scro

fa]

HP

gi|4

7522

826

792.

0(

0.2

6.4(

0.3

2.9(

0.8

10.9

(2.

84.

9(

1.2

6.1(

0.7

<0.0

10.

377

<0.0

1K

038

Tra

nsf

erri

n[S

us

scro

fa]

TF

gi|1

3619

226

20.

3(

0.1

3.7(

0.7

11.0

(2.

42.

1(

0.1

14.7

(1.

019

.8(

1.4

<0.0

1<0

.01

<0.0

1K

089

Tra

nsf

erri

n[S

us

scro

fa]

TF

gi|1

3619

226

1N

D2.

4(

0.6

6.4(

1.7

ND

7.6(

1.0

14.1

(2.

3<0

.01

<0.0

1<0

.05

K04

0T

ran

sfer

rin

[Su

ssc

rofa

]T

Fgi

|136

192

209

0.3(

0.1

3.7(

0.7

11.0

(2.

42.

1(

0.1

14.7

(1.

019

.8(

1.4

<0.0

1<0

.01

<0.0

1K

039

Tra

nsf

erri

n[S

us

scro

fa]

TF

gi|1

3619

220

0N

D2.

4(

0.6

6.4(

1.7

ND

7.6(

1.0

14.1

(2.

3<0

.01

<0.0

1<0

.05

P15

2H

emo

pex

in[S

us

scro

fa]

HP

Xgi

|475

2273

693

ND

1.7(

1.1

9.1(

0.9

ND

6.3(

1.4

13.2

(1.

2<0

.01

<0.0

10.

057

L137

Ret

ino

lb

ind

ing

pro

tein

2,ce

llula

r[S

us

scro

fa]

RB

P2

gi|5

5741

711

389

19.4

(1.

311

4.3(

9.2

108.

6(

6.7

74.4

(10

.411

5.3(

10.4

157.

5(

6.7

<0.0

1<0

.01

<0.0

5L1

38R

etin

ol

bin

din

gp

rote

in2,

cellu

lar

[Su

ssc

rofa

]R

BP

2gi

|557

4171

142

913

.4(

1.3

103.

1(

9.2

107.

5(

2.5

53.3

(3.

911

6.5(

10.3

163.

0(

3.8

<0.0

1<0

.01

<0.0

5

Cel

lR

edo

xH

om

eost

asis

K04

9B

eta-

glo

bin

[Su

ssc

rofa

]B

Ggi

|120

5644

5513

531

.2(

4.5

31.3

(5.

911

2.7(

22.8

60.0

(2.

686

.6(

9.0

230.

9(

28.3

<0.0

1<0

.01

<0.0

5P

036

Per

oxi

red

oxi

n-5

[Su

ssc

rofa

]P

RD

X5

gi|4

7523

086

118

1.6(

0.2

28.5

(7.

131

.2(

3.6

1.5(

0.3

9.5(

1.0

13.0

(1.

1<0

.01

<0.0

1<0

.05

K08

8P

ero

xire

do

xin

-1[B

osta

uru

s]P

RD

X1

gi|6

6773

956

157

3.3(

0.5

7.2(

0.2

8.7(

0.6

3.2(

0.6

3.6(

0.1

8.2(

1.1

<0.0

5<0

.01

<0.0

5K

090

PR

ED

ICT

ED

:si

mila

rto

Ch

lori

de

intr

acel

lula

rch

ann

elp

rote

in1

[Can

isfa

mil

iari

z]C

LIC

1gi

|739

7342

276

1.8(

0.1

9.3(

2.4

13.2

(2.

41.

5(

0.1

4.1(

1.7

11.1

(2.

2<0

.05

<0.0

10.

404

L090

PR

ED

ICT

ED

:si

mila

rto

Per

oxi

red

oxi

n-6

[Su

ssc

rofa

]P

RD

X6

gi|1

9404

2134

759.

2(

0.5

ND

ND

4.9(

1.0

ND

ND

<0.0

1<0

.01

<0.0

1

P00

6P

rote

ind

isu

lfid

eis

om

eras

e-as

soci

ated

3[O

ryct

olag

us

cun

icu

lus]

PD

IA3

gi|2

2687

5264

281

3.2(

0.8

8.8(

0.4

6.2(

0.7

3.5(

0.7

9.6(

1.5

13.2

(1.

8<0

.05

<0.0

01<0

.05

Stre

ssR

esp

on

seP

157

Glu

tath

ion

eS-

tran

sfer

ase

om

ega

[Su

ssc

rofa

]G

STO

gi|4

7522

916

180

3.3(

1.1

39.5

(9.

046

.4(

5.8

2.8(

0.5

20.1

(2.

922

.4(

1.1

<0.0

1<0

.01

0.05

7P

153

Hea

tsh

ock

70kD

ap

rote

in8

[Bos

tau

rus]

HSP

A8

gi|2

2569

8069

245

2.5(

0.6

6.8(

0.1

7.2(

0.5

1.5(

0.2

4.1(

1.3

1.8(

0.7

<0.0

1<0

.01

<0.0

5L1

81H

eat

sho

ckco

gnat

e71

kDa

pro

tein

[Hom

osa

pie

ns]

HSC

70gi

|242

3468

691

4.3(

0.9

3.3(

0.8

1.3(

0.1

2.6(

0.1

1.5(

0.1

0.7(

0.1

<0.0

1<0

.01

0.47

3

K02

194

kDa

glu

cose

-reg

ula

ted

pro

tein

[Su

ssc

rofa

]G

RP

94gi

|475

2301

628

72.

1(

0.4

9.2(

1.1

10.4

(2.

01.

9(

0.5

2.4(

0.4

6.7(

1.7

<0.0

1<0

.01

0.05

5

Cel

lula

rA

po

pto

sis

L024

Cat

hep

sin

B[S

us

scro

fa]

CT

SBgi

|171

9487

7674

35.8

(1.

93.

3(

0.4

2.7(

0.4

14.3

(2.

53.

2(

0.6

2.6(

0.8

<0.0

1<0

.01

<0.0

1L0

78P

RE

DIC

TE

D:

sim

ilar

toLe

uko

cyte

elas

tase

inh

ibit

or

[Su

ssc

rofa

]LE

Igi

|194

0379

5133

71.

7(

0.2

12.6

(1.

818

.5(

2.9

6.8(

0.2

10.4

(2.

117

.6(

2.3

<0.0

5<0

.01

0.15

5

P15

6P

RE

DIC

TE

D:

sim

ilar

to14

-3-3

pro

tein

epsi

lon

[Can

isfa

mil

iari

z]14

-3-

3Egi

|674

6442

416

1N

D3.

5(

1.8

10.4

(2.

35.

8(

0.9

ND

3.2(

0.2

<0.0

5<0

.01

<0.0

1

L037

Cal

reti

culin

[Ory

ctol

agu

scu

nic

ulu

s]C

ALR

gi|1

2672

3562

759.

2(

1.2

ND

ND

2.8(

0.3

ND

ND

<0.0

1<0

.01

<0.0

1L0

34P

RE

DIC

TE

D:

sim

ilar

toca

lret

icu

linis

ofo

rm2

[Can

isfa

mil

iari

z]C

ALR

gi|7

3986

460

168

4.4(

0.8

13.4

(1.

26.

0(

1.4

2.6(

0.3

4.1(

1.1

5.0(

0.3

<0.0

1<0

.01

<0.0

1

aSp

ot

nu

mb

ers

refe

rto

pro

tein

spo

tn

um

ber

sth

atco

rres

po

nd

toth

ela

bel

sin

Fig

ure

s2

and

3.b

Pro

tein

sco

rege

ner

ated

by

MS

iden

tifi

cati

on

pla

tfo

rm,

wit

ha

sco

reo

ver

71b

ein

gco

nsi

der

edas

stat

isti

cal

sign

ifica

nce

.c

Pro

tein

exp

ress

ion

qu

anti

ty,

exp

ress

edas

Mea

n(

SEM

,n

)3

gels

for

bo

thIU

GR

and

NB

Wgr

ou

ps

atea

chag

e.A

llth

eid

enti

fied

spo

tsw

ere

dif

fere

nti

ally

exp

ress

ed(P

<0.

05).

dN

D,

pro

tein

spo

tsw

ere

no

td

etec

tab

le.



Table 5. Cellular Location and Functions of Proteins Differentially Expressed in the Jejunal Mucosa of IUGR Piglets during theSuckling Period

spotno.a protein name subcellular location functions

L162 Albumin [Sus scrofa] Extracellular space; protein complex Transport (DNA, protein, fattyacid, copper, pyridoxalphosphate)

L161L041K081P020 PREDICTED: similar to clathrin, light polypeptide isoform

A [Canis familiariz]Clathrin coat of trans-Golgi networkvesicle

Vesicle-mediated transport

L2101 Chymodenin [Sus scrofa domestica] Secreted; extracellular space DigestionL113 Chymotrypsinogen B [Bos taurus] Secreted; extracellular space DigestionK074 Ezrin [Bos taurus] (Villin-2) Microvillus membrane Regulation of cell shapeK054 COFILIN protein [Sus scrofa] Cytoplasm Cytoskeleton orgnizationL1368 Gamma-actin [Homo sapiens] Cellular structure Tight junctionK082 PREDICTED: similar to Keratin 8 [Sus scrofa] Cellular structure Structural molecule activityK073 PREDICTED: similar to Villin 1 [Sus scrofa] Cytoplasm Cytoskeleton orgnizationL1862 Creatine kinase [Canis familiariz ]/[Sus scrofa] Cytoplasm Creatine pathwayK100K099P027 Cytochrome b5 [Oryctolagus cuniculus] Endoplasmic reticulum membrane Electron transportP111 Cytochrome c oxidase subunit 5B, mitochondrial [Sus

scrofa]Mitochondrion inner membrane Oxidative phosphorylation

L1415 Cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] Cytoplasm GlycerophospholipidmetabolismL099

K084 Enolase 1 [Bos taurus] Cytoplasm Glycolysis/gluconeogenesisK087 Fructose-bisphosphate aldolase A [Bos taurus] Cytoplasm Glycolysis/gluconeogenesisL075 Glycerol-3-phosphate dehydrogenase 1-like protein

(GPD1L protein) [Bos taurus]Cytoplasm Glycerophospholipid

metabolismL061 Mitochondrial succinate dehydrogenase complex subunit

A [Sus scrofa]Mitochondrion Tricarboxylic acid cycle

L074 Triosephosphate isomerase [Sus scrofa] Cytoplasm Carbohydrate metabolismL094 Apolipoprotein A-I [Sus scrofa] Secreted; high-density lipoprotein

particleLipid transport

P013L1146 Apolipoprotein A-IV [Sus scrofa] Secreted; high-density lipoprotein

particleLipid transport

P121 Fatty acid binding protein 5 [Sus scrofa] Cytoplasm Lipid bindingL049 Fatty acid-binding protein [Sus scrofa] Cytoplasm Lipid bindingK101 3-hydroxyanthranilate 3,4-dioxygenase [Bos taurus] Cytoplasm Tryptophan metabolismK085 Aminoacylase I [Sus scrofa] Cytoplasm Urea cycle and metabolism of

amino groupsK076 Mitochondrial ornithine aminotransferase [Sus scrofa] Mitochondrion Arginine and proline

metabolismL006 S-adenosylhomocysteine hydrolase [Sus scrofa] Cytoplasm Methionine metabolismL189 Haptoglobin [Sus scrofa] Secreted; extracellular region Iron transportK038 Transferrin [Sus scrofa] Secreted; extracellular region Iron transportK089K040K039P152 Hemopexin [Sus scrofa] Secreted; extracellular region Iron transportL137 Retinol binding protein 2, cellular [Sus scrofa] Cytoplasm Retinal transportL138K049 Beta-globin [Sus scrofa] Hemoglobin complex Oxygen transportP036 Peroxiredoxin-5 [Sus scrofa] Cytoplasm Cell redox homeostasisK088 Peroxiredoxin-1 [Bos taurus] Cytoplasm Cell redox homeostasisK090 PREDICTED: similar to Chloride intracellular channel

protein 1 [Canis familiariz]Brush border Redox regulation

L090 PREDICTED: similar to Peroxiredoxin-6 [Sus scrofa] Cytoplasm Cell redox homeostasisP006 Protein disulfide isomerase-associated 3 [Oryctolagus

cuniculus]Endoplasmic reticulum Protein disulfide

isomerizationP157 Glutathione S-transferase omega [Sus scrofa] Cytoplasm Stress response; glutathione

transferase activityP153 Heat shock 70 kDa protein 8 [Bos taurus] Cytoplasm, nucleus Stress responseL181 Heat shock cognate 71 kDa protein [Homo sapiens] Cytoplasm, nucleus Stress responseK021 94 kDa glucose-regulated protein [Sus scrofa] Endoplasmic reticulum lumen Stress responseL024 Cathepsin B [Sus scrofa] Cytoplasm ProteolysisL078 PREDICTED: similar to Leukocyte elastase inhibitor [Sus

scrofa]Cytoplasm Protease inhibitor

P156 PREDICTED: similar to 14-3-3 protein epsilon [Canisfamiliariz]

Cytoplasm Cell growth and death

L037 Calreticulin [Oryctolagus cuniculus] Endoplasmic reticulum lumen Cell cycle arrestL034 PREDICTED: similar to calreticulin isoform 2 [Canis

familiariz]Endoplasmic reticulum lumen Cell cycle arrest

a Spot numbers refer to protein spot numbers that correspond to the labels in Figures 2 and 3.



was increased (P < 0.05) in the IUGR jejunum at D7 and D21,respectively, compared with NBW piglets.

Cellular Apoptosis. Four proteins related to apoptosis weredifferentially altered in IUGR piglets. Levels of leukocyteelastase inhibitor (LEI, Spot L078) were reduced (P < 0.05) butthose of cathepsin B (CTSB, Spot L024) were increased (P <0.05) at D1 in the IUGR jejunum, compared with NBW piglets.Abundance of calreticulin (CALR, Spots L034, L037) was higher(P < 0.05) at D1 in IUGR than in NBW piglets. In contrast, levelsof 14-3-3 protein epsilon (14-3-3E, Spot P156) in the IUGRgroup were elevated (P < 0.05) at D7 and D21 but down-regulated in the IUGR group at D1, compared with NWB piglets.

Validation of Proteomic Data by Western Blotting. Figure4 shows the Western blot analysis of 4 proteins (ALB, APOA1,GRP94 and PRDX1) randomly selected from Table 4 forvalidation of proteomic data. The Western blotting results areconsistent with the findings from the temporal proteomicsanalysis.

Discussion

IUGR predisposes offspring to malfunction and delayeddevelopment of the small intestine.3,4,18 Our previous study hasshown a marked alteration of the jejunal proteome in IUGRpiglets at birth.5 The present investigation extended this workto 1- to 21-day-old IUGR piglets reared by sows and furtheridentified 56 differentially expressed jejunal proteins that arerelated to intestinal growth, development and health. Specif-

ically, these proteins affect (1) absorption, digestion andtransport of nutrients; (2) cell structure and motility; (3) glucoseand energy metabolism; (4) lipid metabolism; (5) amino acidmetabolism; (6) mineral and vitamin metabolism; (7) cell redoxhomeostasis; (8) stress response; and (9) cellular apoptosis.Interestingly, our results indicated that IUGR did not affecteither levels of growth factor receptors for gut development orthe abundance of signaling components responsible for intes-tinal growth. It is possible that these proteins were expressedat low levels in the intestinal mucosa of young pigs and thatthere was a limit for their detection by the 2-DE proteomictechnology. To our knowledge, results of the current researchindicate, for the first time, postnatal changes of the intestinalproteome in IUGR neonates.

Absorption, Digestion and Transport. Albumin (ALB) servesas a transporter of lipid-soluble molecules and certain minerals(e.g., steroid hormones, bile salts, unconjugated bilirubin, freefatty acids, calcium, and iron) in blood circulation. ALB in thejejunal mucosa of preweaning piglets is derived primarily fromthe ingested milk. The reduced levels of this protein in thejejunal mucosa of IUGR piglets throughout the suckling periodmay result from malabsorption of macromolecules and mal-nutrition, which can contribute to enteropathy and edema(Figure 1, C1). Additionally, low levels of chymotrypsinogen B(CTRB; a precursor of the digestive enzyme chymotrypsin) andchymodenin (a peptide that specifically stimulates chymot-rypsinogen secretion)19 between D1 and D21 likely impairs

Figure 2. Representative temporal protein expression profiles in the jejunum of IUGR (A-C) and NBW (D-F) piglets at Day 1 (A andD), Day 7 (B and E) and Day 21 (C and F) of life.



digestion ability in the IUGR jejunum. Furthermore, clathrin(light polypeptide isoform A; CLTA) plays a vital role in thegeneration of vesicles for transport of nutrients, antigens,growth factors, pathogens and recycling receptors in endocy-tosis.20 This protein is also required for the function of themitotic spindle in cellular mitosis.21 Thus, continuous reductionin jejunal expression of CLTA may further compromise intra-

cellular signaling cascade, as well as absorption, digestion andtransport ability in the small intestine of IUGR offspring.

Cell Structure and Motility. The 5 proteins, ezrin (EZR), villin1 (VIL1), gamma-actin (ACTG1), COFILIN protein (CFL1), andkeratin 8 (KRT8) play important roles in cell structure andmotility. EZR (also known as villin 2; VIL2) and VIL1, whichare microvillar proteins in intestinal epithelial cells, are requiredfor cell surface adhesion, migration, and organization,22 therebyinvolved in intestinal absorption.23 Moreover, VIL1 is a calcium-regulated, actin binding protein that is vital for establishmentof the apical microvilli in the intestinal brush border.24 Notably,ACTG1 is a highly conserved protein involved in various typesof cell motility, and maintenance of the cytoskeleton, such astight junction.25 Likewise, CFL1 is the major component ofintranuclear and cytoplasmic actin rods which bind to G-actinand F-actin, thereby recycling older ADP-F-actin, helping thecell to maintain the ATP-G-actin pool for sustained motility.26

In addition, KRT8 functions in maintaining the cell structureas the major intermediate filament protein in the intestinalepithelia. The reduction of EZR, VIL1, ACTG1, CFL1, KRT8 in1- to 21-day-old IUGR offspring may result in structuralabnormality and atrophy of their intestinal villus (Figure 1).

Glucose and Energy Metabolism. Glucose is utilized for ATPproduction by the piglet small intestine and is the major sourceof NADPH for synthetic processes.27 An important finding ofthis study is that IUGR reduced expression of key enzymesinvolved in glucose and energy metabolism in jejunum. Forexample, reduced levels of enolase 1 (ENO1), fructose-bispho-sphate aldolase A (FBPA) and triosephosphate isomerase (TPI1)may impair glycolysis in the IUGR intestine. Furthermore,reduced expression of a number of proteins in the Krebs cycle(e.g., succinate dehydrogenase complex subunit A; SDHA) andthe cytochrome C system [e.g., cytochrome b5 (CYB5) andcytochrome c oxidase subunit 5B (COX5B)] may further impairoxidation of energy substrates and ATP production by the IUGRjejunum.

Figure 3. Abundance of temporal differentially expressed proteinsin the jejunum of IUGR and NBW piglets at D1, D7 and D21 oflife (A), as well as their functional classification (B).

Figure 4. Western blotting analysis of jejunal mucosal proteins,ALB (A), APOA1 (B), GRP94 (C) and PRDX1 (D). Data are mean (SEM; n ) 6 for each group at D1, D7, and D21. *, P < 0.05 versusthe IUGR group at each age.



Lipid Metabolism. Lipid is the major source of energy forsuckling piglets. Additionally, milk-borne cholesterol is crucialfor development of the brain and insulin-sensitive tissues ofneonates.28 Apolipoprotein A-I (APOA1) and apolipoproteinA-IV (APOA4) are major component apoproteins in chylomi-crons and high-density lipoproteins (HDL).29 The former is a28 kDa protein that vitally participates in cholesterol transportfrom extrahepatic tissues to the liver.30 In addition, FABP5 andFABP1, which belong to the fatty-acid binding protein (FABP)family, are responsible for intracellular transport of free fattyacids (FFAs) to specific metabolic pathways for utilization.31

Thus, APOA1, APOA4, fatty acid binding protein 5 (FABP5), andfatty acid-binding protein 1 (FABP1) play important roles inlipid metabolism. Consequently, reduced expression of theseproteins in the IUGR jejunum will impair neonatal survival anddevelopment.

Amino Acid Metabolism. The 4 proteins, 3-hydroxyanthra-nilate 3,4-dioxygenase (3HAO), aminoacylase I (ACY1), mito-chondrial ornithine aminotransferase (OAT) and S-adenosyl-homocysteine hydrolase (AHCY), play important role in aminoacid metabolism. 3HAO is an enzyme that employs iron as acofactor to catalyze 3-hydroxyanthranilate coupled with O2 toproduce 2-amino-3-carboxymuconate semialdehyde,32 therebyparticipating in tryptophan metabolism. AHCY is an enzymewhich converts S-adenosylhomocysteine to homocysteine inthe transsulfuration pathway for methionine degradation.Although quantitatively there is little oxidation of tryptophanand methionine to CO2 in enterocytes of neonatal pigs,33

physiological metabolites produced by 3HAO and AHCY (e.g.,hydrogen sulfide) may be important for development of thegut immune system34 and gaseous signaling.35

ACY1 is an enzyme in the family of hydrolases, whichcatalyzes the hydrolysis of N-acyl-L-amino acids to carboxylateand L-amino acid, thereby participating in metabolism of thearginine family of amino acids.2 Specifically, the availability ofN-acetylglutamate plays an important role in the synthesis ofarginine (an essential amino acid) by enterocytes.10 ACY1catalyzes the hydrolysis of N-acetyl-ornithine (derived frommicrobial metabolism in the intestinal lumen) to ornithine,which is subsequently converted into pyrroline-5-carboxylateby OAT or putrescine by ornithine decarboxylase.10 Thereduced expression of both ACY1 and OAT in the jujenalmucosa of IUGR offspring may impair arginine and polyaminesynthesis and, therefore, whole-body arginine homeostasis andgrowth.

Mineral and Vitamin Metabolism. Transferrin (TF), he-mopexin (HPX) and haptoglobin (HP) play a vital role in ironmetabolism. TF (an iron binding transport protein) is respon-sible for iron transport from sites of absorption and hemedegradation to those of storage and utilization.36 The continu-ous reduction of TF in the jejunal mucosa of IUGR pigletsbetween D1 and D21 of life may result in iron deficiency.Additionally, HPX and HP are acute phase proteins. HPX isresponsible for binding heme and transporting it to the liverfor breakdown and iron recovery. This aids in controlling heme-iron availability in peripheral tissues by returning to thecirculation in the form of free HPX.37 HP is required forhomeostasis of ferroportin (a transmembrane protein foundon the surface of cells that stores or transports iron out of cells),thus, contributing to the regulation of iron transfer fromintestinal mucosa to plasma.38 The consistently low levels ofHPX on D7 and D21 in the jejunal mucosa of IUGR piglets is

expected to further compromise iron availability in the body.These novel findings help explain iron deficiency in IUGRoffspring.39

Retinol binding protein 2 (RBP2) is the sole retinol (vitaminA) transporter in the small intestinal epithelium.40 Vitamin Ais a fat-soluble vitamin necessary for growth, reproduction,differentiation of epithelial tissues, and vision.41 The reducedlevel of RBP2 in the IUGR jejunum during the entire sucklingperiod will result in vitamin A deficiency, thereby impairingnumerous retinol-dependent pathways and intestinal devel-opmemnt in young mammals.41

Cell Redox Homeostasis. Beta-globin (BG), protein disulfideisomerase-associated 3 (PDIA3), peroxiredoxin-1 (PRDX1), per-oxiredoxin-5 (PRDX5), peroxiredoxin-6 (PRDX6) and chlorideintracellular channel protein 1 (CLIC1) play critical role incellular redox homeostasis. BG (also known as HBB), along withalpha globin (HBA), is the major component of hemoglobin(an oxygen transporter).42 The continuous reduction of BGbetween D1 and D21 of life may contribute to a defect inoxygen transport and, therefore, intestinal ischemia that oftenoccurs in IUGR neonates (including human infants).43

PDIA3 (also known as ERp57) is a thiol oxidoreductasemember of the protein disulfide isomerase (PDI) family whoseprimary function is protein disulfide isomerization.44 Recentstudies have shown that PDIA3 is redox-active center for controlof intestinal transport.45 The reduction of PDLA3 in the IUGRintestine suggests that its redox-sensitive signaling pathwaysmay be attenuated. PRDX1, PRDX5 and PRDX6, involved incellular redox regulation, belong to the ubiquitous family ofantioxidant enzymes that play important roles in eliminatingperoxides generated during metabolism.46 In response tooxidative stress and/or apoptosis, these enzymes are overex-pressed to protect cells against oxidative stress and/or apoptosisby directly eliminating hydrogen peroxide (H2O2) and neutral-izing other reactive oxygen species (ROS).47 Furthermore,CLIC1, which is localized on the plasma membrane, regulateskey metabolic processes involving stabilization of cell mem-brane potential, transepithelial transport, maintenance ofintracellular pH, and regulation of cell volume.48 Up-regulationof CLIC1 often occurs in response to oxidative stress.49 Col-lectively, the overexpression of PRDX1, PRDX5 and PRDX6, aswell as CLIC1, indicates a redox imbalance in the smallintestinal mucosa of IUGR offspring.

Stress Response. Heat shock 70 kDa protein 8/heat shockcognate 71 kDa protein (HSPA8/HSC70), 94 kDa glucose-regulated protein (GRP94) and glutathione S-transferase omega(GSTO) play a vital role in cellular stress response. HSPA8 (alsoknown as HSC70) and GRP94 are heat shock proteins (HSP),whose expression is increased in response to elevated temper-atures or oxidative stress.50 GSTO, which is a component ofthe glutathione-ascorbate cycle, regulates the activities ofglutathione-dependent thiol transferase and dehydroascorbatereductase as part of antioxidant metabolism and cellular redoxhomeostasis.8,51 The increases in HSPA8, GRP94 and GSTO inthe IUGR jejunum provide another line of evidence for thepresence of oxidative stress during the postnatal life.

Cellular Apoptosis. Cathepsin B (CTSB), calreticulin (CALR),leukocyte elastase inhibitor (LEI) and 14-3-3 protein epsilon(14-3-3E) participate in cellular apoptosis. CTSB is an enzymaticprotein belonging to the peptidase or protease families.52 CTSBcontributes to cell apoptosis by promoting proteolytic cascadeto generate apoptotic peptides.53 CALR is a multifunctionalprotein localized in storage compartments associated with the



endoplasmic reticulum.54 Emerging evidence shows that a highlevel of CALR induces caspase-dependent immunogenic apo-ptosis in cells.55 Additionally, 14-3-3E is a highly conservedacidic protein that belongs to the 14-3-3 protein family andpromotes apoptosis.56 Furthermore, this protein inhibits cellsproliferation and differentiation by inactivating Raf-157 andmitogen-activated protein kinase-1 (BMK1).58 Thus, the eleva-tion of 14-3-3E in the jejunum of IUGR piglets may hinder thegrowth and maturation of their gut.

Conclusion

In summary, results of this study provide the first evidencefor an alteration of the proteome in the small intestine of IUGRoffspring, predisposing the gut to multiple metabolic defectsduring the neonatal period. Specifically, our findings revealincreased levels of proteins related to oxidative stress andapoptosis, as well as decreased abundance of proteins requiredfor digestion, absorption and metabolism of nutrients (includ-ing glucose, lipids, amino acids, vitamins and minerals).Collectively, these changes may be the major mechanismsresponsible for intestinal growth arrest, atrophy and dysfunc-tion in IUGR neonates.

Acknowledgment. This work was supported by theNatural Science Foundation of China (no. u0731001,30810103902), Beijing Natural Science Foundation (no.6082017), the Program for New Century Excellent Talents inUniversity (no. NCET-08-0530), National Research InitiativeCompetitive Grant (No. 2008-35203-19120 and2008-35206-18764) from the USDA National Institute ofFood and Agriculture, and Texas AgriLife Research(H-82000).

Supporting Information Available: The protein iden-tification information is available. This material is available freeof charge via the Internet at http://pubs.acs.org.

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