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Genomics, Proteomics, and Metabolomics

Intrauterine Growth Restriction Affects the Proteomes of the Small Intestine, Liver, and Skeletal Muscle in Newborn Pigs^1,2

³ State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100094; ⁴ Department of Animal Science, Texas A&M University, College Station, TX 77843; ⁵ College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan, China 410128; ⁶ Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, China 410128; ⁷ Xiang-Ya Medical School, Central South University, Changsha, Hunan, China 410078; and ⁸ Protein Chemistry Laboratory, Texas A&M University, College Station, TX 77843

^* To whom correspondence should be addressed. E-mail: g-wu{at}tamu.edu, defali{at}public2.bta.net.cn or yinyulong{at}isa.ac.cn.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Efficiency of nutrient utilization is high in neonates with normal birth weights but is reduced in those with intrauterine growth restriction (IUGR). However, the underlying mechanisms are largely unknown. This study was conducted with the piglet model and proteomics technology to test the hypothesis that IUGR affects expression of key proteins that regulate growth and development of the small intestine, liver, and muscle, the major organs involved in the digestion, absorption, and metabolism of dietary nutrients. Jejunum, liver, and gastrocnemius muscle were obtained from IUGR and normal birth-weight piglets at birth for analysis of proteomes using the 2-dimensional-PAGE MS technology. The results indicate that IUGR decreased the levels of proteins that regulate immune function (immunoglobulins and annexin A1), oxidative defense (peroxiredoxin 1, transferrin, and -crystallin), intermediary metabolism (creatine kinase, alcohol dehydrogenase, L-lactate dehydrogenase, prostaglandin F synthase, apolipoprotein AI, catecho O-methyltransferase, and phosphoglycerate kinase 1), protein synthesis (eukaryotic translation initiation factor-3), and tissue growth (β-actin, desmin, and keratin 10) in a tissue-specific manner. In addition, IUGR increased the levels of proteins that are involved in proteolysis (proteasome -5 and -1 subunits), response to oxidative stress (scavenger-receptor protein and -1 acid glycoprotein), and ATP hydrolysis (F1-ATPase). These novel findings suggest that cellular signaling defects, redox imbalance, reduced protein synthesis, and enhanced proteolysis may be the major mechanisms responsible for abnormal absorption and metabolism of nutrients, as well as reduced growth and impaired development of the small intestine, liver, and muscle in IUGR neonates.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Intestine, muscle, and liver are major organs involved in the digestion, absorption, and metabolism of dietary nutrients (8). Interestingly, small intestine and skeletal muscle are disproportionately reduced and their functions are severely impaired in IUGR piglets (7). Additionally, emerging evidence from studies with rats highlights metabolic defects in skeletal muscle and liver of IUGR progeny (9,10). However, the underlying mechanisms for abnormal nutrient utilization in IUGR neonates are largely unknown.

Proteomics technologies facilitate the simultaneous analyses of thousands of proteins in a tissue, therefore providing useful tools for discovery research in nutritional sciences (11,12). We hypothesized that IUGR affects expression of key proteins that regulate growth and development of the small intestine, liver, and skeletal muscle. This hypothesis was tested with the newborn piglet, a widely used animal model for studying physiology and nutrition of the human infant (4,5,13).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
This work was performed with the approval of Texas A&M University's Institutional Animal Use and Care Committee.

    Chemicals. Protease inhibitors were obtained from Roche Applied Sciences. DNase and RNase were purchased from Worthington. Unless indicated, all other chemicals were procured from Sigma Chemicals.

    Pigs and tissue collection. Pregnant gilts were fed 2 kg/d of a corn- and soybean meal-based diet and had free access to drinking water, as we previously described (14). At term birth (d 114 of gestation), 1 IUGR piglet and 1 NBW piglet were obtained from each of 8 litters. Newborn piglets were killed by jugular puncture after anesthesia, as previously described (15). The small intestine in the neonatal pig was defined as the portion of the digestive tract between the pylorus and the ileocecal valve, with the first 10-cm segment being duodenum. The jejunum constituted 40% of the small intestine below the duodenum and the ileum constituted 60%. The whole small intestine, including mid-jejunum (the middle portion of the jejunum), liver, and gastrocnemius muscle were rapidly dissected and weighed (16). Samples (5 g) of each tissue were immediately placed in liquid nitrogen and stored at –80°C.

    Preparation of tissues for proteomics analysis. Four IUGR and 4 NBW piglets were selected randomly from 8 piglets of each group for the analysis of tissue proteomes. Frozen jejunum, liver, and muscle samples (50 mg) were crushed with mortar and pestle in liquid nitrogen. These powders were dissolved in 0.4 mL of the ice-cold lysis buffer (8 mol/L urea, 1% 3-[3-(-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 30 mmol/L Tris, pH 8.5) containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 5 mg/L leupeptin, 5 mg/L aprotinin, 5 mg/L pepstatin, and 5 mg/L bestatin), placed in ice, and mixed every 5 min for 15 min (30 s per vortex) (17). Four microliters of a nuclease solution (2.37 units of DNAse/µL and 4.49 units of RNAse/µL in 0.5 mol/L Tris buffer and 100 µmol/L MgCl₂) was then added to the homogenate followed by thorough mixing. The homogenate was centrifuged at 12,000 x g; 15 min and 4°C. The supernatant fluid was obtained and stored at –80°C. Its protein concentrations were determined in 1:200 dilution using the microBCA method (18). The presence of urea in the tissue extracts (40 mmol/L) did not interfere with the protein assays.

    Difference gel electrophoresis and MS analysis. These procedures were performed in duplicate. Fifty micrograms of proteins from each sample were labeled with Cy5 or Cy3 (Amersham Biosciences). Switched labeling was applied for each sample as an experimental duplicate. The internal reference, consisting of 400 µg protein (a mixture of 50 µg protein from each of 8 samples) that was labeled with Cy2, was divided into 8 equal portions for loading to gels. A differentially labeled sample for each gel contained an IUGR piglet sample (50 µg protein), a NBW piglet sample (50 µg protein), and an internal reference (50 µg protein). All the samples were subjected to 2-dimensional (2D)-PAGE in the dark, as we previously described (18). The difference gel electrophoresis (DIGE) gels were scanned using Typhoon (Amersham Biosciences), as described by Lilley (19). The DeCyder software 6.5 (Amersham Biosciences) was used for image analysis (20). Changes in 2D-DIGE images were aligned with Coomassie-stained protein patterns in preparative gels and protein spots of interest were obtained manually (18). Gel pieces were digested with 3 µL of 10 µg/L trypsin, as we previously described (18). The peptide fragments produced from each protein spot were used to generate peptide mass fragment data via matrix-assisted laser desorption/ionization-time of flight MS analysis (Bruker Reflex Daltonik). A saturated solution of -cyano-4-hydroxycinnamic acid (prepared in 50% acetonitrile and 0.1% trifluoroacetic acid) was used as a matrix. One microliter of the matrix solution and 1 µL of sample solution were mixed and loaded onto the Score384 target well. The parameters used for the mass fragment data via matrix-assisted laser desorption/ionization-time of flight mass analysis were 20 kV accelerating voltage and 23 kV reflecting voltage at the reflectron-mode setting. The MS analysis generated a mass spectrum of the peptide mixture, which included posttranslational modifications of proteins.

    Protein identification. The peptide mass fingerprinting analysis was performed using the Mascot search engine of Matrix Science (21). Search parameters included: 1) the type of search: peptide mass fingerprint; 2) enzyme of protein digestion: trypsin; 3) mass values: monoisotopic; 4) peptide mass: unrestricted; 5) peptide mass tolerance: ± 0.3 Da; 6) maximum missed cleavages: 1; and 7) variable modifications: carbamidomethylation of cysteine and oxidation of methionine (22). Modifications of cysteine and methionine were selected because dithiothreitol and iodoacetamide were used between the 1st and 2nd dimension of electrophoresis (18). For protein identification by the peptide mass fingerprinting method, peptide masses were searched against the MS protein sequence database for other mammals (23). Mascot computed the probability for the observed match between the experimental data and mass values calculated from a candidate protein or peptide sequence. A significance threshold was set at 0.05; the probability of the observed event occurring by chance was <1 in 20. The software reported a score, which is –10Log₁₀(P), where P is the probability. In this study, a significant match (P 0.05) is a score of 56. The Mascot search also generated values of sequence coverage, which is the percentage of trypsin-derived peptides accounting for the whole sequence of the target protein.

    Statistical analysis. Data are expressed as means ± SEM. Results were statistically analyzed by the t test, using the software provided by Amersham Biosciences. P 0.05 was considered significant.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Body and tissue weights of newborn piglets. In this study, IUGR piglets had a 40% lower (P < 0.01) body weight than NBW piglets (Table 1). The relative weights of both gastrocnemius muscle and small intestine were reduced (P < 0.05) in IUGR piglets compared with NBW piglets. Although the absolute weight of the liver was lower (P < 0.01) in IUGR than in NBW piglets, its relative weight did not differ between these 2 groups (Table 1). Concentrations of protein in the liver, gastrocnemius muscle, and small intestine did not differ between IUGR and NBW piglets and the values (% of wet tissue weight) were 13.1 ± 0.17, 9.15 ± 0.11, and 14.8 ± 0.14 (n = 16), respectively.

View larger version (98K): [in this window] [in a new window]   FIGURE 1  Location and abundance of 11 differentially expressed protein spots in the small intestine of newborn piglets with IUGR and NBW. Frozen jejunum sample (50 mg) was homogenized in Tris buffer containing protease inhibitors. After treatment with DNase and RNase, tissue homogenates (50 µg protein each from an IUGR sample, an NBW sample, and a mixed internal standard) were subjected to fluorescence-based 2D-DIGE. The protein spots in the Cy2 reference DIGE gel that exhibited changes are indicated as S1–S11.

View larger version (124K): [in this window] [in a new window]   FIGURE 2  Location and abundance of 9 differentially expressed protein spots in the liver of newborn piglets with IUGR and with NBW. Frozen liver sample (50 mg) was homogenized in Tris buffer containing protease inhibitors. After treatment with DNase and RNase, tissue homogenates (50 µg protein each from an IUGR sample, an NBW sample, and a mixed internal standard) were subjected to fluorescence-based 2D-DIGE. The protein spots in the Cy2 reference DIGE gel that exhibited changes are indicated as L1–L9.

View larger version (104K): [in this window] [in a new window]   FIGURE 3  Location and abundance of 13 differentially expressed protein spots in gastrocnemius muscle of newborn piglets with IUGR and with NBW. Frozen gastrocnemius-muscle sample (50 mg) was homogenized in Tris buffer containing protease inhibitors. After treatment with DNase and RNase, tissue homogenates (50 µg protein each from an IUGR sample, an NBW sample, and a mixed internal standard) were subjected to fluorescence-based 2D-DIGE. The protein spots in the Cy2 reference DIGE gel that exhibited changes are indicated as M1–M13.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Fetal growth restriction is associated with abnormal intestinal structure, metabolism, and function (2). This is manifest as a reduction in intestinal mucosal mass, malabsorption of nutrients, and necrotizing enterocolitis. Using the proteomics analysis, we found that there were 11 differentially expressed proteins in the small intestine of IUGR piglets (Table 2) that are related primarily to energy metabolism, immune response, cellular structure, and antioxidant function. Because albumin and Ig in newborns are derived from pregnant dams through the fetal swallowing of amniotic fluid and may be synthesized in fetal tissues (2), our results suggest that 1 or both of these 2 processes are impaired in response to fetal growth restriction. Consequently, these compromised neonates are more susceptible to infections when they are challenged by food-borne or other environmental pathogens (24). Additionally, because creatine kinase has a central role in energy storage and there is a particularly high demand for ATP by the small intestine (25), low levels of this protein in IUGR piglets may impair creatine-phosphate formation and ATP-dependent pathways, including nutrient transport arginine synthesis, and protein synthesis. Interestingly, relative protein levels for cytoskeletal β-actin (a microfilament), desmin (a member of the type-3 family of intermediate filaments near the Z line in sarcomeres of muscle cells), and scavenger-receptor protein (a protein that binds acetylated or oxidized low-density lipoprotein) increased in the gut of IUGR piglets compared with NBW piglets (Table 2). These results may be explained by decreased concentrations of nonstructural proteins and also suggest the presence of intestinal oxidative stress in IUGR piglets.

Liver plays a major role in the metabolism of nutrients and other dietary substances (8). Increasing evidence suggests defects in both metabolic regulation and physiological function of IUGR livers (10,26). Consistent with these findings, we identified 9 differentially expressed proteins in the liver of IUGR piglets that play important roles in energy and iron metabolism, protein degradation, and cellular signal transduction (Table 3). Transferrins are iron-binding transport proteins that are responsible for the transport of iron from sites of absorption and heme degradation to those of storage and utilization (27). A reduced availability of transferrins in the liver of IUGR neonates may result in iron deficiency, thereby impairing numerous iron-dependent pathways. Indeed, IUGR infants are at an increasing risk for anemia, but the underlying mechanisms have not yet been elucidated (27). In addition, because L-lactate dehydrogenase, alcohol dehydrogenase, and -crystalline (an NADPH:quinine oxidoreductase) catalyze NAD⁺- or NADP⁺-dependent reactions, low concentrations of these enzymes in the liver of IUGR piglets may alter cellular redox state, H⁺ concentrations, lactate/pyruvate metabolism, and ethanol detoxification. Notably, phosphatidylethanolamine-binding protein inhibits mitogen-activated protein kinase pathway and phosphatidylethanolamine externalization (28), whereas proteasome (a large multi-subunit protease complex) plays a key role in protein degradation (29). Increased concentrations of these 2 proteins, along with prostaglandin F synthase, may alter cellular signaling, the metabolism of nutrients (particularly fatty acids), and protein accretion in the liver. Also, elevated levels of -1 acid glycoprotein (an acute phase protein synthesized by the liver) suggest systemic oxidative stress in IUGR neonates. These effects will negatively impact substrate utilization, nutrient homeostasis, immune response, and hepatic function (24,30).

Skeletal muscle normally represents 35–40% of the body weight in newborns. Interestingly, natural or experimentally induced IUGR is associated with impaired growth and development of this tissue (2,7). Reduced myofiber number in IUGR neonates limits the capacity for postnatal compensatory growth of skeletal muscle (31). There is evidence that IUGR affects the composition of muscle and the distribution of muscle fiber type, as well as fat and collagen concentrations in skeletal muscle (32,33). In support of these previous findings, we detected changes in the levels of 12 proteins that are related primarily to macronutrient metabolism (particularly protein synthesis and degradation), immune response, cellular structure, extracellular matrix, and antioxidant function (Table 4). Particularly, the levels of cellular structural proteins [keratin 10 (a type-1 intermediate filament synthesized by keratinocytes) and β-actin] were decreased in gastrocnemius muscle of IUGR piglets (Table 4). Also, concentrations of catechol O-methyltransferase [an enzyme responsible for the degradation of catecholamine neurotransmitters and hormones (e.g. dopamine, epinephrine, and norepinephrine)] were reduced in IUGR muscle, which may play a role in the maladjustment of IUGR neonates to the extrauterine life (2). Furthermore, intramuscular levels for proteins involved in fat metabolism (apolipoprotein AI), protein synthesis (eukaryotic translation initiation factor-3), cellular redox balance, and signal transduction (peroxiredoxin 1 and annexin A1) were also lower in IUGR piglets. In contrast, concentrations of proteasome were markedly elevated in skeletal muscle of IUGR piglets (Table 4), therefore enhancing ubiquitin-dependent protein degradation (34). In addition, concentrations of mitochondrial F1-ATPase increased in IUGR muscle (Table 4), which would accelerate ATP hydrolysis and reduce its availability for ATP-dependent pathways, including nutrient transport and protein synthesis in skeletal muscle. When protein synthesis is reduced, amino acids are directed to oxidation and fatty acid synthesis (8). Thus, these changes are expected to decrease protein accretion and increase fat deposition in skeletal muscle of IUGR piglets (33,34).

The relative weights of gastrocnemius muscle and small intestine were 21 and 14% lower in IUGR than in NBW piglets, respectively (Table 1). The disproportionate reductions of quadriceps muscle and small intestine in newborn IUGR piglets were also reported by Widdowson (7). The weights of other tissues were not determined in this study. However, results of published work indicate that the relative weights of the brain, heart, stomach, and large intestine were 21, 17, 10, and 9.5% greater, respectively, in IUGR than in NBW newborn piglets (35). In addition, whole-body water content was 2.7% higher in newborn IUGR piglets than in NBW piglets (7). Because concentrations of proteins in gastrocnemius muscle, small intestine, and liver did not differ between IUGR and NBW piglets, the total amounts of specific proteins that exhibited lower concentrations in these tissues (expressed per 50 µg of a protein mixture) were decreased further in IUGR neonates. Additionally, because all the tissues consist of a heterogeneous population of cells that may differ between IUGR and NBW neonates (36), future studies are necessary to determine cell-specific changes of proteomes in response to fetal growth restriction. Also, caution should be exercised to interpret data on proteins in the interstitial matrix, which include albumin precursor in the small intestine and skeletal muscle as well as keratin 10 in the muscle (Tables 2 and 4). Changes in concentrations of these proteins may result from alterations in the extracellular space of mucosal and muscle cells (37,38). However, it should be borne in mind that extracellular proteins are not only essential components of tissue growth but also play an important role in the metabolism and physiology of hepatocytes, enterocytes, and myofibers (8,9,39).

In conclusion, IUGR affects the proteomes of the small intestine, liver, and skeletal muscle in newborn piglets primarily in a tissue-specific manner. These diverse proteins are known to regulate cellular signaling, immune response, oxidative defense, and intermediary metabolism. Thus, the altered expression of the proteomes is expected to increase proteolysis, reduce polypeptide synthesis, induce oxidative stress, and compromise health in IUGR neonates. This may be the major mechanisms responsible for abnormal absorption and metabolism of nutrients, as well as reduced growth and impaired development of the small intestine, liver, and muscle in IUGR piglets. Our findings provide a new framework for studying the impact of IUGR on the proteomes of other mammals.

	ACKNOWLEDGMENTS

 
We thank Scott Jobgen, Wenjuan Jobgen, and Elena Lyuksyutova for technical assistance and Frances Mutscher for manuscript preparation.

	FOOTNOTES

 
¹ Supported by the National Basic Research Program of China (no. 2004CB117503), National Natural Science Foundation of China (no. 30121004, 30525029, 30528006, and 30600434), Outstanding Overseas Chinese Scholar Fund of the Chinese Academy of Sciences (no. 2005-1-4), Beijing Municipal Natural Science Foundation (no. 6082017), NIEHS P30-ES09106, and the Texas Agricultural Experiment Station (no. H-8200).

² Author disclosures: J. J. Wang, L. X. Chen, D. F. Li, Y. L. Yin, X. Q. Wang, P. Li, L. J. Dangott, W. X. Hu, and G. Wu, no conflicts of interest.

⁹ Abbreviations used: 2D, 2-dimensional; DIGE, difference gel electrophoresis; Ig, immunoglobulin; IUGR, intrauterine growth restriction; NBW, normal birth weight.

Manuscript received 30 August 2007. Initial review completed 19 September 2007. Revision accepted 11 October 2007.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, J. Articles by Wu, G. Search for Related Content PubMed PubMed Citation Articles by Wang, J. Articles by Wu, G. Pubmed/NCBI databases Medline Plus Health Information High Risk Pregnancy