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Intrauterine growth restriction alters the hepatic proteome in fetal pigs
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Journal of Nutritional Biochemistry 24 (2013) 954 – 959
Intrauterine growth restriction alters the hepatic proteome in fetal pigs☆ Chuang Liu a , Gang Lin a , Xiaoqiu Wang a , Taiji Wang a , Guoyao Wu a, b , Defa Li a , Junjun Wang a,⁎ b
a State Key Laboratory of Animal Nutrition, China Agricultural University, 100193 Beijing, China Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, TX 77843, USA
Received 25 January 2011; received in revised form 7 June 2012; accepted 20 June 2012
Abstract Intrauterine growth restriction (IUGR) is a major problem in both humans and animals. The IUGR fetus has abnormal metabolism of nutrients in the liver. This study was conducted with comparative proteomic approach and biochemical analyses to test the hypothesis that IUGR alters the hepatic proteome in the fetal liver. Livers were obtained from IUGR and normal-weight fetal pigs at Day 110 of gestation. Twenty-two differentially expressed proteins in the liver were identified between IUGR and normal fetal pigs. These proteins participate in the intermediary metabolism of nutrients (including glucose, amino acids, protein, lipids, vitamins and minerals), oxidative stress, as well as cell structure and growth. Of particular interest, the IUGR fetus had a higher activity of glutamate oxaloacetate transaminase and a lower activity of lipoprotein lipase than the normal ones. These results indicate altered metabolism of nutrients, abnormal ammonia utilization, and reduced capacity for detoxification in the liver of IUGR fetus. Collectively, the findings have important implication for explaining low food efficiency and understanding the mechanism responsible for impaired growth in IUGR neonates. © 2013 Elsevier Inc. All rights reserved. Keywords: Fetus; Intrauterine growth restriction; Liver; Pigs; Proteome
1. Introduction Intrauterine growth restriction (IUGR) is a major problem in human medicine [1] and animal production [2]. IUGR can be defined as impaired growth and development of the mammalian embryo/ fetus or its organs during pregnancy [2], which can be measured as fetal or birth weight less than 2 standard deviations of the mean body weight for gestational age [3]. In the IUGR fetus, the liver (a major organ in nutrient utilization [4]) exhibits a plethora of abnormalities in metabolism, development and function [2,5]. For example, increasing evidences showed derangements in both mitochondrial oxidative phosphorylation and energy metabolism of the livers of IUGR offspring [6,7]. Additionally, our previous work indicated that IUGR affected expression of hepatic proteins that were related to the metabolism of energy, iron and protein in newborn neonates [8]. Fetal programming can permanently impact growth and health of offspring [2]. Thus, prenatal interventions provide an attractive means to ameliorate and prevent metabolic defects in neonates and ☆
This work was supported by the National Basic Research Program of China (No. 2012CB124703), the National Natural Science Foundation of China (No. 30972156 and 31129006) the Thousand-People Talent program at China Agricultural University, the Key Project of Chinese Ministry of Education (No. 104240), and National Research Initiative Competitive Grant (No. 200835203-19120 and 2008-35206-18764) from the USDA National Institute of Food and Agriculture. ⁎ Corresponding author. Tel.: +86 10 62733588; fax: +86 10 62733688. E-mail address: [email protected] (J. Wang). 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.06.016
adults. However, this targeted approach should be based, in part, on specific alterations of protein expression in the fetus. At present, such knowledge is limited in the literature. We hypothesized that IUGR alters the hepatic proteome in the fetal liver. The present study was conducted with the pig (an excellent animal model to study human nutrition and metabolism [9]) using comparative proteomic approach and biochemical analyses [9,10]. 2. Materials and methods 2.1. Pigs and tissue collection During pregnancy, six primiparous Dalland dams were fed 2 kg/d of a corn and soybean meal-based diet and had free access to drinking water. At day 110 of gestation, pigs were killed by jugular puncture after anesthesia, as we described previously [8]. The whole uterus was removed carefully, and 1 IUGR fetus and 1 normal-weight fetus were obtained from each of six sows. The blood on the left lobe of liver was rapidly flushed out with saline then rapidly placed in liquid nitrogen and stored at −80°C. 2.2. Protein extraction from the liver Liver samples from four IUGR and four normal fetuses were used for protein extraction, as we described previously [3]. Briefly, frozen liver samples (approximately 50 mg) were crushed to powder with mortar and pestle in liquid nitrogen. These powder were solubilized in 400 μL lysis buffer (7 M Solid Urea, 2 M Thiourea, 4% CHAPS, and 15 mM Tris, pH 8.5) containing 1% protease inhibitor (100×) (GE Healthcare, Piscataway, NJ, USA), put in ice, and mixed every 5 min for 3 times. Use an Ultrasonicater Model VCX 500 (Sonics & Materials, Newtown, CT, USA) at 20% power output for 10 min with 2-s on and 8-s off cycles to break up the mixture at 0°C. Add 1% (v/v) nuclease mix (100×) (GE Healthcare), then the lysed cell suspension was kept at room temperature for 1 h to dissolve proteins, followed by resonication as described above to thoroughly rupture cell membranes [11]. The homogenate was subsequently
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Table 1 Biochemical properties of differentially expressed spots in liver of IUGR fetal pigs No.
Accession
Name
Abbr.
Ratios for IUGR to normal fetal pigs
Score
P value
Carbamoylphosphate synthase I [Homo sapiens] Similar to carbamoylphosphate synthase [ammonia], mitochondrial [Sus scrofa] Similar to carbamoylphosphate synthase [ammonia], mitochondrial [Sus scrofa] Thimet oligopeptidase [Sus scrofa] Phosphoglucomutase 1 [Sus scrofa] Similar to glutamate dehydrogenase 1, partial [Sus scrofa] Inositol-3-phosphate synthase 1 isoform 3 [Homo sapiens] Inositol-3-phosphate synthase 1 isoform 1 [Homo sapiens] 6-phosphogluconate dehydrogenase (249 AA) [Sus scrofa] Similar to fumarylacetoacetate hydrolase (fumarylacetoacetase) [Sus scrofa] 4-hydroxyphenylpyruvate dioxygenase [Sus scrofa]
CPS1 CPS1 CPS1 THOP PGM1 GDH1 IPS1 IPS1 6GPD FAH HPD
−1.24 −1.27 −1.30 −1.20 −1.24 +1.24 +1.44 +1.30 −1.21 −1.24 +1.24
91 117 117 189 124 101 99 117 74 84 101
.029 .034 .005 .029 .019 .036 .037 .049 .036 .024 .034
Vitamin and mineral metabolism L5 CAQ34904 Transferrin [Sus scrofa] L10 Q29550 Retinyl ester hydrolase [Sus scrofa] L18 NP_002892 Reticulocalbin-1 precursor [Homo sapiens] L24 NP_001138695 Cellular retinoic acid binding protein 1 [Sus scrofa]
TF REH RCN1 CRABP1
−1.22 −1.47 +1.20 −1.35
77 131 115 134
.027 .011 .016 .007
Oxidative stress L12 CAO81735 L21 NP_999331 L22 AAA30983 L23 NP_999554 L25 AAX62515
Alternative pig liver esterase [Sus scrofa] Dimeric dihydrodiol dehydrogenase [Sus scrofa] Alpha-1 acid glycoprotein[Sus scrofa] Glutathione S-transferase alpha M14 [Sus scrofa] Liver fatty acid binding protein [Sus scrofa]
APLE DD AGP GSTAM14 L-FABP
−1.56 −1.25 +1.43 −2.86 +1.64
190 106 98 111 116
.016 .046 .008 .014 .045
Cell structure and growth L4 XP_001926037 L7 NP_004514 L11 AF357236.1 L14 BAA11928 L20 AAS55927
Similar to ribosome receptor [Sus scrofa] Kinesin-like protein KIF11 [Homo sapiens] Polytrophin [Homo sapiens] ER-60 protease [Homo sapiens] Cytoskeletal beta actin [Sus scrofa]
RR KIF11 TROPH ER-60 β-actin
−1.20 −1.23 +1.65 +1.45 +1.32
373 76 87 97 102
.039 .025 .010 .047 .044
Intermediary metabolism L1 BAD74207 L2 XP_001926358 L3 XP_001926358 L6 NP_999388 L8 AAC64130 L9 XP_001925088 L13 NP_001164410 L15 NP_057452 L16 CAA34633 L17 XP_001928326 L19 NP_999389
Livers were obtained from IUGR and normal-weight fetal pigs on Day 110 of pregnancy. After flushing away blood with saline, the left lobe of the liver was rapidly placed in liquid nitrogen and stored at −80°C. The ratios of protein levels in the liver of IUGR fetal pigs to these for normal-weight fetal pigs were calculated. The twenty-five spots indicated in the table were differentially expressed (Pb.05). The signs (−) and (+) indicate a decrease and increase, respectively, compared with the value for normal-weight fetal pigs. Score of those spots generated by MS identification platform, with a score over 72 is considered as statistical significance.
centrifuged at 13,000×g; 10 min and 4°C. The supernatant fluid was obtained, and its protein concentration was determined using the Bradford method. The aliquots of liver protein were stored at −80°C. 2.3. Difference gel electrophoresis and mass spectrometry analysis Proteins in each group were fluorescently labeled with Cy2, Cy3 and Cy5 (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's protocol for minimal labeling. In order to avoid any dye-specific labeling artifacts, two samples of each group were labeled with Cy3 and the other two with Cy5 (400 pmol dye/50 μg protein). The internal reference, a mixture of 25 μg protein from each of 8 samples (consisting of 200 μg protein), was labeled with Cy2. A differentially labeled sample for each gel contained an IUGR sample (50 μg protein, Cy3- or Cy5- labeled), a normal sample (50 μg protein, Cy5- or Cy3- labeled), and an internal reference (50 μg protein). A total of 4 gels were run for the two-dimensional fluorescence difference gel electrophoresis through isoelectric focusing and standard vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%), both in the dark, as we previously described [12]. The gels were imaged using Typhoon 9400 laser scanner (GE Healthcare), as described by Lilley [13]. Images were analyzed using the DeCyder version 6.5 software (GE Healthcare) [14]. Differentially expressed protein spots (Pb.05) with a deviation of over 1.2-fold were selected. The difference gel electrophoresis (DIGE) gels were aligned with Coomassie-stained 2D gels and the protein spots of interest were obtained manually for in-gel digestion, as we previously described [8]. MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) was then conducted to identify the peptides from in-gel digested proteins [8]. For the proteins that could not be identified from the Sus scrofa database solely based on peptide mass fingerprinting, further MALDI-TOF/TOF MS was conducted. Considering an incomplete database for porcine proteins, protein homologs identified from the database of Homo sapiens through MS/MS analysis were also included in this study [11].
missed cleavages: 1; (4) taxonomy: other mammalian; (5) fixed modifications: none selected; (6) variable modifications: carbamidomethyl (C) and oxidation (M); (7) peptide mass: unrestricted; (8) peptide mass tolerance: ±0.03–0.29 Da; (9) mass values: MH+ and monoisotopic. In this analysis, a score of ≥72 was considered as significant (Pb.05). 2.5. Western blotting Extracted proteins (30 μg) were resolved by electrophoresis (Bio-Rad, Richmond, CA) on a 12.5% SDS-PAGE. The proteins were transferred electrophoretically to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with TBST (0.05% Tween 20, 100 mM Tris–HCl and 150 mM NaCl, pH 7.5) containing 5% fat-free dry milk at 4°C overnight, the membranes were incubated with primary antibodies, which were antiGAPDH, anti-GDH1, anti-CPS1 anti-L-FABP and anti-transferrin (Santa Cruz Biotechnology, Heidelberg, Germany) in dilution of 1:2000, 1:2000, 1:2000, 1:1000 and 1:1000, respectively for 2 h; rinsed the membranes adequately in TBST and incubated them with a secondary antibody (horseradish peroxidase-labeled anti-rabbit IgG and anti-mouse IgG diluted in 1:2000) for 1 h. After incubation with a chemiluminescence substrate, the protein bands were visualized using a gel-imaging system (Tanon Science and Technology, Shanghai, China) with Image Analysis Software (National Institutes of Health, Bethesda, MD, USA). 2.6. Biochemical analysis Liver homogenate (1% and 10%, 0.86% ice-cold physiological saline as medium) were conducted manually using a glass tissue grinder. The activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, hepatic lipase and lipoprotein lipase in both IUGR and normal fetal livers were measured using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). One unit of activity for hepatic lipase, and lipoprotein lipase was defined as production of 1 μmol free fatty acid/h in 1 mL of assay solution.
2.4. Protein identification 2.7. Statistical analysis The peptide mass fingerprinting analyses of MS and MS/MS data were performed using the Mascot search engine of Matrix Science (http://www.matrixscience.com). Search parameters included: (1) database: NCBInr; (2) enzyme: trypsin; (3) maximum
Data were statistically analyzed by the t test, using SAS (version 8.2; SAS Institute, Cary, NC, USA). Pb.05 was considered as significant.
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3. Results 3.1. Differentially expressed proteins A total of 25 protein spots were differentially expressed in the liver between IUGR and normal fetuses. Their biochemical information is summarized in Table 1. Fig. 1 and Fig. 2 show their appearances and abundances on the gel images. According to their biological functions, these proteins can be divided into several groups: (1) intermediary metabolism; (2) vitamin and mineral metabolism; (3) oxidative stress and (4) cell structure and growth.
3.1.1. Proteins involved in intermediary metabolism Eleven spots of proteins were related to intermediary metabolism. Carbamoylphosphate synthase 1 (Spot L1, L2, L3), thimet oligopeptidase (Spot L6), phosphoglucomutase 1 (Spot L8), 6-phosphogluconate dehydrogenase (Spot L16) and fumarylacetoacetate hydrolase (Spot L17) were down-regulated in the liver of IUGR fetuses, when compared with normal fetuses. In contrast, glutamate dehydrogenase 1 (Spot L9), inositol-3-phosphate synthase 1 (Spot L13, L15) and 4hydroxyphenylpyruvate dioxygenase (Spot L19) were up-regulated in the IUGR group.
3.1.2. Proteins involved in vitamin and mineral metabolism Differentially expressed proteins that are known to play roles in vitamin and mineral metabolism include: transferrin (Spot L5), retinyl ester hydrolase (Spot L10), reticulocalbin-1 precursor (Spot L18) and cellular retinoic acid binding protein 1 (Spot L24). In comparison with normal fetuses, except for reticulocalbin-1 precursor whose abundance was increased, other proteins were reduced in the liver of IUGR fetuses.
3.1.3. Proteins involved in oxidative stress Compared with normal fetuses, alpha-1 acid glycoprotein (Spot L22) and liver fatty acid binding protein (L-FABP, Spot L25) were upregulated in the liver of IUGR fetuses. Alternative porcine liver esterase (Spot L12), dimeric dihydrodiol dehydrogenase (Spot L21) and glutathione S-transferase alpha M14 (Spot L23) were downregulated. These five proteins were related to oxidative stress. 3.1.4. Proteins involved in cell structure and growth Expression of five liver proteins related to cell functions was affected by IUGR. Specifically, ribosome receptor (Spot L4) and kinesin-like protein KIF11 (KIF11, Spot L7) were reduced in the liver of IUGR fetuses, compared with the normal fetuses. In contrast, polytrophin (Spot L11), ER-60 protease (Spot L14) and cytoskeletal beta actin (Spot L20) were increased in the liver of IUGR group, compared with the normal group. 3.2. Validation of proteomic data by Western blotting Fig. 3 shows the data on Western blotting analysis of four proteins (glutamate dehydrogenase 1, carbamoylphosphate synthase 1, L-FABP and transferrin) selected from Table 1 for validation of proteomic data. The Western blotting results were consistent with the findings from the comparative proteomics analysis. 3.3. Measurement of activities of key metabolic enzymes The IUGR fetal pig liver had a higher activity of glutamate oxaloacetate transaminase and a lower activity of lipoprotein lipase than the normal fetuses. There were no differences in the hepatic activity of glutamate pyruvate transaminase or hepatic lipase between IUGR and normal groups (Table 2).
Fig. 1. Distribution of differentially expressed hepatic proteins between IUGR and normal-weight fetal pigs in the two-dimensional gel. L1-L3: Carbamoylphosphate synthase 1; L4: Ribosome receptor; L5: Transferrin; L6: Thimet oligopeptidase; L7: kinesin-like protein KIF11; L8: Phosphoglucomutase 1; L9: Glutamate dehydrogenase 1, partial; L10: Retinyl ester hydrolase; L11: Polytrophin; L12: Alternative pig liver esterase; L13, L15: Inositol-3-phosphate synthase 1; L14: ER-60 protease; L16: 6-phosphogluconate dehydrogenase; L17: Fumarylacetoacetate hydrolase; L18: Reticulocalbin-1 precursor; L19: 4-hydroxyphenylpyruvate dioxygenase; L20: Cytoskeletal beta actin; L21: dimeric dihydrodiol dehydrogenase; L22: Alpha-1 acid glycoprotein; L23: Glutathione S-transferase alpha M14; L24: Cellular retinoic acid binding protein 1; L25: Liver fatty acid binding protein.
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Fig. 2. Abundances of differentially expressed proteins in the liver of IUGR and normal-weight fetal pigs at Day 110 of gestation. The letters of single spots on the left side show spot number (L1-L25), and those on the right side show short name. L1-L3: Carbamoylphosphate synthase 1 (CSP1); L4: Ribosome receptor (RR); L5: Transferrin (TF); L6: Thimet oligopeptidase (THOP); L7: kinesin-like protein KIF11 (KIF11); L8: Phosphoglucomutase 1 (PGM1); L9: Glutamate dehydrogenase 1, partia (GDH1); L10: Retinyl ester hydrolase (REH); L11: Polytrophin (TROPH); L12: Alternative pig liver esterase (APLE); L13, L15: Inositol-3-phosphate synthase 1 (ISP1); L14: ER-60 protease (ER-60); L16: 6-phosphogluconate dehydrogenase (6PGD); L17: Fumarylacetoacetate hydrolase (FAH); L18: Reticulocalbin-1 precursor (RCN1); L19: 4-hydroxyphenylpyruvate dioxygenase (HPD); L20: Cytoskeletal beta actin; L21: dimeric dihydrodiol dehydrogenase (DD); L22: Alpha-1 acid glycoprotein (AGP); L23: Glutathione S-transferase alpha M14 (GSTAM14); L24: CRABP1; L25: Liver fatty acid binding protein (L-FABP).
4. Discussion Liver plays a vital role in the absorption and metabolism of nutrients [4]. IUGR impairs the development of the liver and subsequently causes dysfunction of this organ in pigs [2]. At the same time, there is evidence for the low efficiency of food utilization in IUGR piglets [15], but little is known about the underlying mechanism. Our results showed the different proteome and altered activities of key enzymes involved in nutrient metabolism in the liver of IUGR fetal pigs. Specifically, there were 22 differentially expressed proteins which are related to intermediary metabolism of nutrients (including glucose, amino acids, protein, glucose, lipids, vitamins and minerals), oxidative stress, as well as cell structure and growth. These new findings may aid in a better understanding of impaired metabolism in IUGR fetuses. 4.1. Nutrients metabolism Three differentially expressed proteins are related to carbohydrates metabolism, with enhanced expression of inositol-3-phosphate
synthase 1 (EC=5.5.1.4) and reduced expression of phosphoglucomutase 1 (EC=5.4.2.2) and 6-phosphogluconate dehydrogenase (EC=1.1.1.44). Inositol-3-phosphate synthase 1 is a major regulatory enzyme in the synthesis of all inositol-containing compounds (including phosphoinositides and inositol phosphates), and plays an important role in growth regulation, signal transduction, membrane biogenesis and other essential biochemical processes [16,17]. Phosphoglucomutase 1 participates in both glycogenolysis and glycogenesis [18] and 6-phosphogluconate dehydrogenase is the third enzyme of the pentose phosphate pathway for the production of NADPH. Down-regulation of those two proteins may contribute to glycogen accumulation and oxidative stress in the liver of IUGR offspring. Glutamate dehydrogenase 1 (EC=1.4.1.3) is responsible for the generation of ammonia from glutamate, whereas carbamoylphosphate synthase 1 (EC=6.3.4.16), which converts ammonia and bicarbonate into carbamoylphosphate, is a key regulatory enzyme in the hepatic urea cycle [10]. Thus, up-regulated expression of glutamate dehydrogenase 1, coupled with reduced levels of carbamoylphosphate synthase 1, may accelerate ammonia production while impairing the hepatic
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Fig. 3. Western blot analysis of carbamoyl-phosphate synthetase 1 (A), glutamate dehydrogenase 1 (B), liver fatty acid binding protein (C), and transferrin (D) in the liver of IUGR and normal-weight fetal pigs at Day 110 of gestation. Glyceraldehyde-3-phophate dehydrogenase (GADPH) was used as the reference protein. Data are ratio between the expressions in % adjusted volume (Adj. Vol.) of target proteins and the expressions in % adj. vol. of GAPDH, and are mean±S.E.M.; n=4 for each group. *Pb.05 versus the normal group; **Pb.01 versus the normal group.
urea cycle, leading to hyperammonemia in IUGR fetuses [9] and lowbirth-weight infants [19]. The approximately two-fold greater activity of glutamate oxaloacetate transaminase (EC=2.6.1.1) in IUGR fetuses can facilitate the conversion of glutamate and oxaloacetate to aspartate [10], thereby reducing the availability of oxaloacetate for the oxidation of acetyl-CoA via the Krebs cycle. This, in turn, can reduce ATP production and the activation of the urea cycle at the steps of carbamoylphosphate synthase 1 and argininosuccinate synthase, thereby further resulting in elevated levels of ammonia in the blood circulation. The other two down-regulated proteins that participate in amino acid metabolism were 4-hydroxyphenylpyruvate dioxygenase (EC=1.13.11.27) and fumarylacetoacetate hydrolase (EC=3.7.1.2). These enzymes participate in the catabolism of L-phenylalanine and L-tyrosine [20,21]. Lipoprotein lipase hydrolyzes lipids in lipoproteins (in chylomicrons and very low-density lipoproteins) into two free fatty acids and one monoacylglycerol molecule. In the present study, its activity in the liver of runt fetus was reduced, compared with normal ones. This may be a metabolic basis for elevated levels of triglycerides and reduced levels of free fatty acids in the cord serum of IUGR infants [22]. Four proteins were related to vitamin and mineral metabolism. Retinyl ester hydrolase (EC=3.1.1.1) and cellular retinoic acid binding protein type 1 (CRABP1) potentially regulate the release and degradation of retinoic acid. Retinoic acid is one of the derivatives generated from retinol (vitamin A) and is essential for the proliferation and differentiation of a wide variety of cell types [23,24]. The reduced expression of retinyl ester hydrolase and CRABP1 in the liver of IUGR fetuses may impair retinoid and vitamin A homeostasis. Transferrin, a highly conserved serum glycoprotein produced by the liver, plays an important role in iron transport [25]. The lower level of transferrin in the liver of IUGR fetuses may lead to iron deficiency. These results of the proteomic analysis help explain the previous observation that anemia often occurs in IUGR infants [26]. In addition, reticulocalbin-1 precursor is one member of the
proteins in the lumen of the endoplasmic reticulum which bind Ca2+. Increased expression of reticulocalbin-1 precursor in IUGR pigs may decrease efflux of Ca2+ from the endoplasmic reticulum to the cytoplasm, thereby imparing Ca2+-dependent cell signaling. 4.2. Oxidative stress L-FABP has a high capacity for binding peroxidation products of long-chain fatty acids [27] and contains many sulfur amino acid residues (one cysteine and seven methionine residues) [28]. Thus, this protein is a strong endogenous antioxidant [28]. The upregulated level of L-FABP and alpha-1 acid glycoprotein (an acute phase protein) in the IUGR fetuses may indicate that the hepatocytes were suffering from oxidative stress. NADPH is a cofactor for glutathione reductase which converts glutathione disulfide to glutathione, which is an important cellular antioxidant and protects cells against oxidative stress [29]. As noted previously, the pentose phosphate pathway (pentose cycle) produces most of the NADPH in cells. Thus, reduced expression of 6-phosphogluconate dehydrogenase, which catalyzes the conversion of 6-phospho-D-gluconate and NADP+ to D-ribulose 5-phosphate, carbon dioxide and NADPH [30], can exacerbate oxidative stress and dyslipidemia in IUGR offspring. Glutathione S-transferase alpha M14 is a member of the glutathione S-transferase family (EC=2.5.1.18), which catalyzes the Table 2 Activities of hepatic metabolic enzymes in IUGR and normal-weight fetal pigs Glutamate oxaloacetate Glutamate pyruvate Hepatic lipase Lipoprotein transaminase (IU/L) transaminase (IU/L) (U/mL) lipase (U/mL) Normal 1.20±0.31 IUGR 3.13±0.31** P value b.01
0.30±0.05 0.17±0.05 0.11
0.40±0.08 0.27±0.10 0.36
0.77±0.03 0.18±0.06** b.01
Enzyme activities were measured in liver homogenates. Values are means±S.E.M., n=6, **, Pb.01 versus the normal group.
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conjugation of glutathione to electrophiles, thereby detoxifying endogenous toxic substances (e.g., peroxidised lipids) and xenobiotics [31,32]. Xenobiotics are compounds foreign to the organism's normal metabolic process [33]. Xenobiotic metabolism consists of metabolic pathways that modify the chemical structure of these compounds. Alternative pig liver esterase is a novel isoenzyme of liver esterase, which belongs to the class of mammalian carboxylesterases, can also efficiently catalyze the hydrolysis of a variety of ester- and amide-containing substances involved in the hydrolysis of various esters from diet and detoxification of xenobiotics [14,34]. Dimeric dihydrodiol dehydrogenase (EC=1.3.1.20) is one of the enzymes in the metabolism of naphthalene (a kind of xenobiotics). Interestingly, levels of these three proteins were all decreased in the IUGR fetal liver, indicating an impaired capacity for xenobiotic detoxification in runts.
4.3. Cell structure and growth An important finding from this study is that abundances of several key proteins related to cell structure and growth are altered in the liver of IUGR fetuses, including down-regulation of ribosome receptor, kinesin-like protein KIF11 as well as up-regulation of ER-60 protease, polytrophin, and cytoskeletal beta actin. Ribosome receptor is responsible for both ribosome binding and the translocation of nascent proteins across the membrane of the rough ER [35]. Kinesin-like protein KIF11 (KIF11) is a member of kinesin family, which belongs to a class of motor proteins. Down-regulation of this protein can be associated with centrosome movement and cell arrest in mitosis [36]. ER-60 protease plays a role in degrading proteins in the endoplasmic reticulum [37], whereas cytoskeletal beta actin is involved in various types of cell motility. Overexpression of beta actin can alter cell morphology [38] and also affect cell motility [39]. Finally, polytrophin binds beta actin to influence cell structure [38,39]. Thus, low abundances of ribosome receptor and KIF11, coupled with high abundances of ER-60 protease, polytrophin, and cytoskeletal beta actin, may result in abnormal structure, mitosis, morphology, and motility of hepatocytes, thereby impair the functions of the liver in IUGR pigs. In conclusion, IUGR affects the proteome and the activities of key metabolic enzymes in the liver of fetal pigs. The differently expressed proteins are related to the metabolism of glucose, carbohydrates, protein, lipids, amino acids, vitamins, and minerals, as well as oxidative stress, xenobiotic detoxification, and cell functions. These results indicate abnormal metabolism of nutrients and ammonia, reduced antioxidative capacity, and impaired cell growth in the IUGR fetus. The findings have important implications for both human and animal nutrition.
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Journal of Nutritional Biochemistry 24 (2013) 954 – 959
Intrauterine growth restriction alters the hepatic proteome in fetal pigs☆ Chuang Liu a , Gang Lin a , Xiaoqiu Wang a , Taiji Wang a , Guoyao Wu a, b , Defa Li a , Junjun Wang a,⁎ b
a State Key Laboratory of Animal Nutrition, China Agricultural University, 100193 Beijing, China Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, TX 77843, USA
Received 25 January 2011; received in revised form 7 June 2012; accepted 20 June 2012
Abstract Intrauterine growth restriction (IUGR) is a major problem in both humans and animals. The IUGR fetus has abnormal metabolism of nutrients in the liver. This study was conducted with comparative proteomic approach and biochemical analyses to test the hypothesis that IUGR alters the hepatic proteome in the fetal liver. Livers were obtained from IUGR and normal-weight fetal pigs at Day 110 of gestation. Twenty-two differentially expressed proteins in the liver were identified between IUGR and normal fetal pigs. These proteins participate in the intermediary metabolism of nutrients (including glucose, amino acids, protein, lipids, vitamins and minerals), oxidative stress, as well as cell structure and growth. Of particular interest, the IUGR fetus had a higher activity of glutamate oxaloacetate transaminase and a lower activity of lipoprotein lipase than the normal ones. These results indicate altered metabolism of nutrients, abnormal ammonia utilization, and reduced capacity for detoxification in the liver of IUGR fetus. Collectively, the findings have important implication for explaining low food efficiency and understanding the mechanism responsible for impaired growth in IUGR neonates. © 2013 Elsevier Inc. All rights reserved. Keywords: Fetus; Intrauterine growth restriction; Liver; Pigs; Proteome
1. Introduction Intrauterine growth restriction (IUGR) is a major problem in human medicine [1] and animal production [2]. IUGR can be defined as impaired growth and development of the mammalian embryo/ fetus or its organs during pregnancy [2], which can be measured as fetal or birth weight less than 2 standard deviations of the mean body weight for gestational age [3]. In the IUGR fetus, the liver (a major organ in nutrient utilization [4]) exhibits a plethora of abnormalities in metabolism, development and function [2,5]. For example, increasing evidences showed derangements in both mitochondrial oxidative phosphorylation and energy metabolism of the livers of IUGR offspring [6,7]. Additionally, our previous work indicated that IUGR affected expression of hepatic proteins that were related to the metabolism of energy, iron and protein in newborn neonates [8]. Fetal programming can permanently impact growth and health of offspring [2]. Thus, prenatal interventions provide an attractive means to ameliorate and prevent metabolic defects in neonates and ☆
This work was supported by the National Basic Research Program of China (No. 2012CB124703), the National Natural Science Foundation of China (No. 30972156 and 31129006) the Thousand-People Talent program at China Agricultural University, the Key Project of Chinese Ministry of Education (No. 104240), and National Research Initiative Competitive Grant (No. 200835203-19120 and 2008-35206-18764) from the USDA National Institute of Food and Agriculture. ⁎ Corresponding author. Tel.: +86 10 62733588; fax: +86 10 62733688. E-mail address: [email protected] (J. Wang). 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.06.016
adults. However, this targeted approach should be based, in part, on specific alterations of protein expression in the fetus. At present, such knowledge is limited in the literature. We hypothesized that IUGR alters the hepatic proteome in the fetal liver. The present study was conducted with the pig (an excellent animal model to study human nutrition and metabolism [9]) using comparative proteomic approach and biochemical analyses [9,10]. 2. Materials and methods 2.1. Pigs and tissue collection During pregnancy, six primiparous Dalland dams were fed 2 kg/d of a corn and soybean meal-based diet and had free access to drinking water. At day 110 of gestation, pigs were killed by jugular puncture after anesthesia, as we described previously [8]. The whole uterus was removed carefully, and 1 IUGR fetus and 1 normal-weight fetus were obtained from each of six sows. The blood on the left lobe of liver was rapidly flushed out with saline then rapidly placed in liquid nitrogen and stored at −80°C. 2.2. Protein extraction from the liver Liver samples from four IUGR and four normal fetuses were used for protein extraction, as we described previously [3]. Briefly, frozen liver samples (approximately 50 mg) were crushed to powder with mortar and pestle in liquid nitrogen. These powder were solubilized in 400 μL lysis buffer (7 M Solid Urea, 2 M Thiourea, 4% CHAPS, and 15 mM Tris, pH 8.5) containing 1% protease inhibitor (100×) (GE Healthcare, Piscataway, NJ, USA), put in ice, and mixed every 5 min for 3 times. Use an Ultrasonicater Model VCX 500 (Sonics & Materials, Newtown, CT, USA) at 20% power output for 10 min with 2-s on and 8-s off cycles to break up the mixture at 0°C. Add 1% (v/v) nuclease mix (100×) (GE Healthcare), then the lysed cell suspension was kept at room temperature for 1 h to dissolve proteins, followed by resonication as described above to thoroughly rupture cell membranes [11]. The homogenate was subsequently
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Table 1 Biochemical properties of differentially expressed spots in liver of IUGR fetal pigs No.
Accession
Name
Abbr.
Ratios for IUGR to normal fetal pigs
Score
P value
Carbamoylphosphate synthase I [Homo sapiens] Similar to carbamoylphosphate synthase [ammonia], mitochondrial [Sus scrofa] Similar to carbamoylphosphate synthase [ammonia], mitochondrial [Sus scrofa] Thimet oligopeptidase [Sus scrofa] Phosphoglucomutase 1 [Sus scrofa] Similar to glutamate dehydrogenase 1, partial [Sus scrofa] Inositol-3-phosphate synthase 1 isoform 3 [Homo sapiens] Inositol-3-phosphate synthase 1 isoform 1 [Homo sapiens] 6-phosphogluconate dehydrogenase (249 AA) [Sus scrofa] Similar to fumarylacetoacetate hydrolase (fumarylacetoacetase) [Sus scrofa] 4-hydroxyphenylpyruvate dioxygenase [Sus scrofa]
CPS1 CPS1 CPS1 THOP PGM1 GDH1 IPS1 IPS1 6GPD FAH HPD
−1.24 −1.27 −1.30 −1.20 −1.24 +1.24 +1.44 +1.30 −1.21 −1.24 +1.24
91 117 117 189 124 101 99 117 74 84 101
.029 .034 .005 .029 .019 .036 .037 .049 .036 .024 .034
Vitamin and mineral metabolism L5 CAQ34904 Transferrin [Sus scrofa] L10 Q29550 Retinyl ester hydrolase [Sus scrofa] L18 NP_002892 Reticulocalbin-1 precursor [Homo sapiens] L24 NP_001138695 Cellular retinoic acid binding protein 1 [Sus scrofa]
TF REH RCN1 CRABP1
−1.22 −1.47 +1.20 −1.35
77 131 115 134
.027 .011 .016 .007
Oxidative stress L12 CAO81735 L21 NP_999331 L22 AAA30983 L23 NP_999554 L25 AAX62515
Alternative pig liver esterase [Sus scrofa] Dimeric dihydrodiol dehydrogenase [Sus scrofa] Alpha-1 acid glycoprotein[Sus scrofa] Glutathione S-transferase alpha M14 [Sus scrofa] Liver fatty acid binding protein [Sus scrofa]
APLE DD AGP GSTAM14 L-FABP
−1.56 −1.25 +1.43 −2.86 +1.64
190 106 98 111 116
.016 .046 .008 .014 .045
Cell structure and growth L4 XP_001926037 L7 NP_004514 L11 AF357236.1 L14 BAA11928 L20 AAS55927
Similar to ribosome receptor [Sus scrofa] Kinesin-like protein KIF11 [Homo sapiens] Polytrophin [Homo sapiens] ER-60 protease [Homo sapiens] Cytoskeletal beta actin [Sus scrofa]
RR KIF11 TROPH ER-60 β-actin
−1.20 −1.23 +1.65 +1.45 +1.32
373 76 87 97 102
.039 .025 .010 .047 .044
Intermediary metabolism L1 BAD74207 L2 XP_001926358 L3 XP_001926358 L6 NP_999388 L8 AAC64130 L9 XP_001925088 L13 NP_001164410 L15 NP_057452 L16 CAA34633 L17 XP_001928326 L19 NP_999389
Livers were obtained from IUGR and normal-weight fetal pigs on Day 110 of pregnancy. After flushing away blood with saline, the left lobe of the liver was rapidly placed in liquid nitrogen and stored at −80°C. The ratios of protein levels in the liver of IUGR fetal pigs to these for normal-weight fetal pigs were calculated. The twenty-five spots indicated in the table were differentially expressed (Pb.05). The signs (−) and (+) indicate a decrease and increase, respectively, compared with the value for normal-weight fetal pigs. Score of those spots generated by MS identification platform, with a score over 72 is considered as statistical significance.
centrifuged at 13,000×g; 10 min and 4°C. The supernatant fluid was obtained, and its protein concentration was determined using the Bradford method. The aliquots of liver protein were stored at −80°C. 2.3. Difference gel electrophoresis and mass spectrometry analysis Proteins in each group were fluorescently labeled with Cy2, Cy3 and Cy5 (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's protocol for minimal labeling. In order to avoid any dye-specific labeling artifacts, two samples of each group were labeled with Cy3 and the other two with Cy5 (400 pmol dye/50 μg protein). The internal reference, a mixture of 25 μg protein from each of 8 samples (consisting of 200 μg protein), was labeled with Cy2. A differentially labeled sample for each gel contained an IUGR sample (50 μg protein, Cy3- or Cy5- labeled), a normal sample (50 μg protein, Cy5- or Cy3- labeled), and an internal reference (50 μg protein). A total of 4 gels were run for the two-dimensional fluorescence difference gel electrophoresis through isoelectric focusing and standard vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%), both in the dark, as we previously described [12]. The gels were imaged using Typhoon 9400 laser scanner (GE Healthcare), as described by Lilley [13]. Images were analyzed using the DeCyder version 6.5 software (GE Healthcare) [14]. Differentially expressed protein spots (Pb.05) with a deviation of over 1.2-fold were selected. The difference gel electrophoresis (DIGE) gels were aligned with Coomassie-stained 2D gels and the protein spots of interest were obtained manually for in-gel digestion, as we previously described [8]. MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) was then conducted to identify the peptides from in-gel digested proteins [8]. For the proteins that could not be identified from the Sus scrofa database solely based on peptide mass fingerprinting, further MALDI-TOF/TOF MS was conducted. Considering an incomplete database for porcine proteins, protein homologs identified from the database of Homo sapiens through MS/MS analysis were also included in this study [11].
missed cleavages: 1; (4) taxonomy: other mammalian; (5) fixed modifications: none selected; (6) variable modifications: carbamidomethyl (C) and oxidation (M); (7) peptide mass: unrestricted; (8) peptide mass tolerance: ±0.03–0.29 Da; (9) mass values: MH+ and monoisotopic. In this analysis, a score of ≥72 was considered as significant (Pb.05). 2.5. Western blotting Extracted proteins (30 μg) were resolved by electrophoresis (Bio-Rad, Richmond, CA) on a 12.5% SDS-PAGE. The proteins were transferred electrophoretically to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with TBST (0.05% Tween 20, 100 mM Tris–HCl and 150 mM NaCl, pH 7.5) containing 5% fat-free dry milk at 4°C overnight, the membranes were incubated with primary antibodies, which were antiGAPDH, anti-GDH1, anti-CPS1 anti-L-FABP and anti-transferrin (Santa Cruz Biotechnology, Heidelberg, Germany) in dilution of 1:2000, 1:2000, 1:2000, 1:1000 and 1:1000, respectively for 2 h; rinsed the membranes adequately in TBST and incubated them with a secondary antibody (horseradish peroxidase-labeled anti-rabbit IgG and anti-mouse IgG diluted in 1:2000) for 1 h. After incubation with a chemiluminescence substrate, the protein bands were visualized using a gel-imaging system (Tanon Science and Technology, Shanghai, China) with Image Analysis Software (National Institutes of Health, Bethesda, MD, USA). 2.6. Biochemical analysis Liver homogenate (1% and 10%, 0.86% ice-cold physiological saline as medium) were conducted manually using a glass tissue grinder. The activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, hepatic lipase and lipoprotein lipase in both IUGR and normal fetal livers were measured using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). One unit of activity for hepatic lipase, and lipoprotein lipase was defined as production of 1 μmol free fatty acid/h in 1 mL of assay solution.
2.4. Protein identification 2.7. Statistical analysis The peptide mass fingerprinting analyses of MS and MS/MS data were performed using the Mascot search engine of Matrix Science (http://www.matrixscience.com). Search parameters included: (1) database: NCBInr; (2) enzyme: trypsin; (3) maximum
Data were statistically analyzed by the t test, using SAS (version 8.2; SAS Institute, Cary, NC, USA). Pb.05 was considered as significant.
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3. Results 3.1. Differentially expressed proteins A total of 25 protein spots were differentially expressed in the liver between IUGR and normal fetuses. Their biochemical information is summarized in Table 1. Fig. 1 and Fig. 2 show their appearances and abundances on the gel images. According to their biological functions, these proteins can be divided into several groups: (1) intermediary metabolism; (2) vitamin and mineral metabolism; (3) oxidative stress and (4) cell structure and growth.
3.1.1. Proteins involved in intermediary metabolism Eleven spots of proteins were related to intermediary metabolism. Carbamoylphosphate synthase 1 (Spot L1, L2, L3), thimet oligopeptidase (Spot L6), phosphoglucomutase 1 (Spot L8), 6-phosphogluconate dehydrogenase (Spot L16) and fumarylacetoacetate hydrolase (Spot L17) were down-regulated in the liver of IUGR fetuses, when compared with normal fetuses. In contrast, glutamate dehydrogenase 1 (Spot L9), inositol-3-phosphate synthase 1 (Spot L13, L15) and 4hydroxyphenylpyruvate dioxygenase (Spot L19) were up-regulated in the IUGR group.
3.1.2. Proteins involved in vitamin and mineral metabolism Differentially expressed proteins that are known to play roles in vitamin and mineral metabolism include: transferrin (Spot L5), retinyl ester hydrolase (Spot L10), reticulocalbin-1 precursor (Spot L18) and cellular retinoic acid binding protein 1 (Spot L24). In comparison with normal fetuses, except for reticulocalbin-1 precursor whose abundance was increased, other proteins were reduced in the liver of IUGR fetuses.
3.1.3. Proteins involved in oxidative stress Compared with normal fetuses, alpha-1 acid glycoprotein (Spot L22) and liver fatty acid binding protein (L-FABP, Spot L25) were upregulated in the liver of IUGR fetuses. Alternative porcine liver esterase (Spot L12), dimeric dihydrodiol dehydrogenase (Spot L21) and glutathione S-transferase alpha M14 (Spot L23) were downregulated. These five proteins were related to oxidative stress. 3.1.4. Proteins involved in cell structure and growth Expression of five liver proteins related to cell functions was affected by IUGR. Specifically, ribosome receptor (Spot L4) and kinesin-like protein KIF11 (KIF11, Spot L7) were reduced in the liver of IUGR fetuses, compared with the normal fetuses. In contrast, polytrophin (Spot L11), ER-60 protease (Spot L14) and cytoskeletal beta actin (Spot L20) were increased in the liver of IUGR group, compared with the normal group. 3.2. Validation of proteomic data by Western blotting Fig. 3 shows the data on Western blotting analysis of four proteins (glutamate dehydrogenase 1, carbamoylphosphate synthase 1, L-FABP and transferrin) selected from Table 1 for validation of proteomic data. The Western blotting results were consistent with the findings from the comparative proteomics analysis. 3.3. Measurement of activities of key metabolic enzymes The IUGR fetal pig liver had a higher activity of glutamate oxaloacetate transaminase and a lower activity of lipoprotein lipase than the normal fetuses. There were no differences in the hepatic activity of glutamate pyruvate transaminase or hepatic lipase between IUGR and normal groups (Table 2).
Fig. 1. Distribution of differentially expressed hepatic proteins between IUGR and normal-weight fetal pigs in the two-dimensional gel. L1-L3: Carbamoylphosphate synthase 1; L4: Ribosome receptor; L5: Transferrin; L6: Thimet oligopeptidase; L7: kinesin-like protein KIF11; L8: Phosphoglucomutase 1; L9: Glutamate dehydrogenase 1, partial; L10: Retinyl ester hydrolase; L11: Polytrophin; L12: Alternative pig liver esterase; L13, L15: Inositol-3-phosphate synthase 1; L14: ER-60 protease; L16: 6-phosphogluconate dehydrogenase; L17: Fumarylacetoacetate hydrolase; L18: Reticulocalbin-1 precursor; L19: 4-hydroxyphenylpyruvate dioxygenase; L20: Cytoskeletal beta actin; L21: dimeric dihydrodiol dehydrogenase; L22: Alpha-1 acid glycoprotein; L23: Glutathione S-transferase alpha M14; L24: Cellular retinoic acid binding protein 1; L25: Liver fatty acid binding protein.
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Fig. 2. Abundances of differentially expressed proteins in the liver of IUGR and normal-weight fetal pigs at Day 110 of gestation. The letters of single spots on the left side show spot number (L1-L25), and those on the right side show short name. L1-L3: Carbamoylphosphate synthase 1 (CSP1); L4: Ribosome receptor (RR); L5: Transferrin (TF); L6: Thimet oligopeptidase (THOP); L7: kinesin-like protein KIF11 (KIF11); L8: Phosphoglucomutase 1 (PGM1); L9: Glutamate dehydrogenase 1, partia (GDH1); L10: Retinyl ester hydrolase (REH); L11: Polytrophin (TROPH); L12: Alternative pig liver esterase (APLE); L13, L15: Inositol-3-phosphate synthase 1 (ISP1); L14: ER-60 protease (ER-60); L16: 6-phosphogluconate dehydrogenase (6PGD); L17: Fumarylacetoacetate hydrolase (FAH); L18: Reticulocalbin-1 precursor (RCN1); L19: 4-hydroxyphenylpyruvate dioxygenase (HPD); L20: Cytoskeletal beta actin; L21: dimeric dihydrodiol dehydrogenase (DD); L22: Alpha-1 acid glycoprotein (AGP); L23: Glutathione S-transferase alpha M14 (GSTAM14); L24: CRABP1; L25: Liver fatty acid binding protein (L-FABP).
4. Discussion Liver plays a vital role in the absorption and metabolism of nutrients [4]. IUGR impairs the development of the liver and subsequently causes dysfunction of this organ in pigs [2]. At the same time, there is evidence for the low efficiency of food utilization in IUGR piglets [15], but little is known about the underlying mechanism. Our results showed the different proteome and altered activities of key enzymes involved in nutrient metabolism in the liver of IUGR fetal pigs. Specifically, there were 22 differentially expressed proteins which are related to intermediary metabolism of nutrients (including glucose, amino acids, protein, glucose, lipids, vitamins and minerals), oxidative stress, as well as cell structure and growth. These new findings may aid in a better understanding of impaired metabolism in IUGR fetuses. 4.1. Nutrients metabolism Three differentially expressed proteins are related to carbohydrates metabolism, with enhanced expression of inositol-3-phosphate
synthase 1 (EC=5.5.1.4) and reduced expression of phosphoglucomutase 1 (EC=5.4.2.2) and 6-phosphogluconate dehydrogenase (EC=1.1.1.44). Inositol-3-phosphate synthase 1 is a major regulatory enzyme in the synthesis of all inositol-containing compounds (including phosphoinositides and inositol phosphates), and plays an important role in growth regulation, signal transduction, membrane biogenesis and other essential biochemical processes [16,17]. Phosphoglucomutase 1 participates in both glycogenolysis and glycogenesis [18] and 6-phosphogluconate dehydrogenase is the third enzyme of the pentose phosphate pathway for the production of NADPH. Down-regulation of those two proteins may contribute to glycogen accumulation and oxidative stress in the liver of IUGR offspring. Glutamate dehydrogenase 1 (EC=1.4.1.3) is responsible for the generation of ammonia from glutamate, whereas carbamoylphosphate synthase 1 (EC=6.3.4.16), which converts ammonia and bicarbonate into carbamoylphosphate, is a key regulatory enzyme in the hepatic urea cycle [10]. Thus, up-regulated expression of glutamate dehydrogenase 1, coupled with reduced levels of carbamoylphosphate synthase 1, may accelerate ammonia production while impairing the hepatic
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Fig. 3. Western blot analysis of carbamoyl-phosphate synthetase 1 (A), glutamate dehydrogenase 1 (B), liver fatty acid binding protein (C), and transferrin (D) in the liver of IUGR and normal-weight fetal pigs at Day 110 of gestation. Glyceraldehyde-3-phophate dehydrogenase (GADPH) was used as the reference protein. Data are ratio between the expressions in % adjusted volume (Adj. Vol.) of target proteins and the expressions in % adj. vol. of GAPDH, and are mean±S.E.M.; n=4 for each group. *Pb.05 versus the normal group; **Pb.01 versus the normal group.
urea cycle, leading to hyperammonemia in IUGR fetuses [9] and lowbirth-weight infants [19]. The approximately two-fold greater activity of glutamate oxaloacetate transaminase (EC=2.6.1.1) in IUGR fetuses can facilitate the conversion of glutamate and oxaloacetate to aspartate [10], thereby reducing the availability of oxaloacetate for the oxidation of acetyl-CoA via the Krebs cycle. This, in turn, can reduce ATP production and the activation of the urea cycle at the steps of carbamoylphosphate synthase 1 and argininosuccinate synthase, thereby further resulting in elevated levels of ammonia in the blood circulation. The other two down-regulated proteins that participate in amino acid metabolism were 4-hydroxyphenylpyruvate dioxygenase (EC=1.13.11.27) and fumarylacetoacetate hydrolase (EC=3.7.1.2). These enzymes participate in the catabolism of L-phenylalanine and L-tyrosine [20,21]. Lipoprotein lipase hydrolyzes lipids in lipoproteins (in chylomicrons and very low-density lipoproteins) into two free fatty acids and one monoacylglycerol molecule. In the present study, its activity in the liver of runt fetus was reduced, compared with normal ones. This may be a metabolic basis for elevated levels of triglycerides and reduced levels of free fatty acids in the cord serum of IUGR infants [22]. Four proteins were related to vitamin and mineral metabolism. Retinyl ester hydrolase (EC=3.1.1.1) and cellular retinoic acid binding protein type 1 (CRABP1) potentially regulate the release and degradation of retinoic acid. Retinoic acid is one of the derivatives generated from retinol (vitamin A) and is essential for the proliferation and differentiation of a wide variety of cell types [23,24]. The reduced expression of retinyl ester hydrolase and CRABP1 in the liver of IUGR fetuses may impair retinoid and vitamin A homeostasis. Transferrin, a highly conserved serum glycoprotein produced by the liver, plays an important role in iron transport [25]. The lower level of transferrin in the liver of IUGR fetuses may lead to iron deficiency. These results of the proteomic analysis help explain the previous observation that anemia often occurs in IUGR infants [26]. In addition, reticulocalbin-1 precursor is one member of the
proteins in the lumen of the endoplasmic reticulum which bind Ca2+. Increased expression of reticulocalbin-1 precursor in IUGR pigs may decrease efflux of Ca2+ from the endoplasmic reticulum to the cytoplasm, thereby imparing Ca2+-dependent cell signaling. 4.2. Oxidative stress L-FABP has a high capacity for binding peroxidation products of long-chain fatty acids [27] and contains many sulfur amino acid residues (one cysteine and seven methionine residues) [28]. Thus, this protein is a strong endogenous antioxidant [28]. The upregulated level of L-FABP and alpha-1 acid glycoprotein (an acute phase protein) in the IUGR fetuses may indicate that the hepatocytes were suffering from oxidative stress. NADPH is a cofactor for glutathione reductase which converts glutathione disulfide to glutathione, which is an important cellular antioxidant and protects cells against oxidative stress [29]. As noted previously, the pentose phosphate pathway (pentose cycle) produces most of the NADPH in cells. Thus, reduced expression of 6-phosphogluconate dehydrogenase, which catalyzes the conversion of 6-phospho-D-gluconate and NADP+ to D-ribulose 5-phosphate, carbon dioxide and NADPH [30], can exacerbate oxidative stress and dyslipidemia in IUGR offspring. Glutathione S-transferase alpha M14 is a member of the glutathione S-transferase family (EC=2.5.1.18), which catalyzes the Table 2 Activities of hepatic metabolic enzymes in IUGR and normal-weight fetal pigs Glutamate oxaloacetate Glutamate pyruvate Hepatic lipase Lipoprotein transaminase (IU/L) transaminase (IU/L) (U/mL) lipase (U/mL) Normal 1.20±0.31 IUGR 3.13±0.31** P value b.01
0.30±0.05 0.17±0.05 0.11
0.40±0.08 0.27±0.10 0.36
0.77±0.03 0.18±0.06** b.01
Enzyme activities were measured in liver homogenates. Values are means±S.E.M., n=6, **, Pb.01 versus the normal group.
C. Liu et al. / Journal of Nutritional Biochemistry 24 (2013) 954–959
conjugation of glutathione to electrophiles, thereby detoxifying endogenous toxic substances (e.g., peroxidised lipids) and xenobiotics [31,32]. Xenobiotics are compounds foreign to the organism's normal metabolic process [33]. Xenobiotic metabolism consists of metabolic pathways that modify the chemical structure of these compounds. Alternative pig liver esterase is a novel isoenzyme of liver esterase, which belongs to the class of mammalian carboxylesterases, can also efficiently catalyze the hydrolysis of a variety of ester- and amide-containing substances involved in the hydrolysis of various esters from diet and detoxification of xenobiotics [14,34]. Dimeric dihydrodiol dehydrogenase (EC=1.3.1.20) is one of the enzymes in the metabolism of naphthalene (a kind of xenobiotics). Interestingly, levels of these three proteins were all decreased in the IUGR fetal liver, indicating an impaired capacity for xenobiotic detoxification in runts.
4.3. Cell structure and growth An important finding from this study is that abundances of several key proteins related to cell structure and growth are altered in the liver of IUGR fetuses, including down-regulation of ribosome receptor, kinesin-like protein KIF11 as well as up-regulation of ER-60 protease, polytrophin, and cytoskeletal beta actin. Ribosome receptor is responsible for both ribosome binding and the translocation of nascent proteins across the membrane of the rough ER [35]. Kinesin-like protein KIF11 (KIF11) is a member of kinesin family, which belongs to a class of motor proteins. Down-regulation of this protein can be associated with centrosome movement and cell arrest in mitosis [36]. ER-60 protease plays a role in degrading proteins in the endoplasmic reticulum [37], whereas cytoskeletal beta actin is involved in various types of cell motility. Overexpression of beta actin can alter cell morphology [38] and also affect cell motility [39]. Finally, polytrophin binds beta actin to influence cell structure [38,39]. Thus, low abundances of ribosome receptor and KIF11, coupled with high abundances of ER-60 protease, polytrophin, and cytoskeletal beta actin, may result in abnormal structure, mitosis, morphology, and motility of hepatocytes, thereby impair the functions of the liver in IUGR pigs. In conclusion, IUGR affects the proteome and the activities of key metabolic enzymes in the liver of fetal pigs. The differently expressed proteins are related to the metabolism of glucose, carbohydrates, protein, lipids, amino acids, vitamins, and minerals, as well as oxidative stress, xenobiotic detoxification, and cell functions. These results indicate abnormal metabolism of nutrients and ammonia, reduced antioxidative capacity, and impaired cell growth in the IUGR fetus. The findings have important implications for both human and animal nutrition.
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