Preterm neonates born before 37 weeks of gestation have an intestine that is structurally and functionally immature, including a low digestive capacity, dysmotility, and impaired barrier function (1). All of these defects may render preterm neonates vulnerable to feeding-induced disorders, detrimental immune responses (2,3), and necrotizing enterocolitis (NEC) (1). Regardless, the intestine of preterm newborns may show a remarkable feeding-induced increase in some functions (4,5), although it is unknown how these responses differ from those in term newborns. Furthermore, it is not known which intestinal defects predispose preterm neonates to NEC (2).
We hypothesized that the intestinal trophic response to enteral formula feeding is similar in preterm and term neonates, as indicated by changes in intestinal mass and activities of brush border enzymes. In addition, we hypothesized that the elevated sensitivity to NEC-like lesions in the immature intestine would be reflected in changes of the intestinal proteome, and thus reflects changes in a range of intestinal functions related to ontogenetic development. Preterm pigs have already been used extensively to study neonatal intestinal development (4,6), and after enteral feeding of both preterm and term pigs for 1 day, we compared the expression of intestinal proteins using gel-based proteomics. Gel-based proteomic technology allows a large-scale screening of tissue proteins and has been previously used to detect changes in intestinal proteins related to enteral feeding, microbial colonization, antibiotics, and NEC development in preterm pigs (7–10). The proteins and pathways identified in the present study may help to explain the specific role of prematurity, and how gestational age at birth may influence intestinal proteins and, possibly, the high sensitivity to feeding-induced gut disorders in preterm infants.
Animals and Their Treatment
Under anesthesia, 47 preterm pigs were obtained from 14 pregnant sows (Large White × Landrace × Duroc; Askelygaard, Denmark) by cesarean delivery at 90% to 92% (105–107 days) gestation, as described earlier (5). Eleven of these pigs were not fed and euthanized within 6 hours of birth (sodium pentobarbitone, 200 mg/kg, intravenous). The intestine was dissected out and the wet weight and length were recorded. Small sections of the small intestine were excised at 50% along the length of the intestine (middle intestine), frozen at −80oC, and used for later determinations of activities of disaccharidase (sucrase, maltase, lactase) and peptidase (aminopeptidase A and N, dipeptidylpeptidase IV), as described previously (5). A 10-cm section of the excised middle intestine was used for calculating the proportion of mucosa after scraping off the mucosa part with a plastic slide and drying both mucosa and muscularis layers overnight in an oven to determine mass on a dry matter basis.
The remaining 36 preterm pigs were reared individually in infant incubators (Air-Shields, Hatboro, PN) and fitted with orogastric catheters and fed with infant formula (15 mL/kg every 3 hours), as described previously (4). The infant formula used was designed to match the macronutrient composition of sow's milk during lactation (4,11) and contained 80 g peptide, 70 g Maxipro, and 75 mL Liquigen-MCT mixed with 1 L water (all products kindly donated by Nutricia, Birkerød, Denmark). After being fed for 24 to 36 hours, all pigs were euthanized and tissues were collected as described above for newborn preterm pigs. For these pigs, we also recorded the NEC incidence according to our macroscopic NEC evaluation system, in which 1 = absence of lesions; 2 = local hyperemia, inflammation, and edema; 3 = hyperemia, extensive edema, and local hemorrhage; 4 = extensive hemorrhage; 5 = local necrosis and pneumatosis intestinalis; and 6 = extensive necrosis and pneumatosis intestinalis. NEC diagnosis was defined as a pig with a score of minimum 3 in minimum 1 intestinal region (proximal, middle, distal intestine, colon).
Term pigs (n = 28) were obtained from 5 pregnant sows (Large White × Landrace × Duroc, Askelygaard, Denmark) and delivered close to full term (115–116 days’ gestation, either by cesarean section or after spontaneous vaginal birth). Ten newborn, unfed pigs were euthanized within 6 hours of birth, whereas the other 18 piglets were reared in infant incubators, fed formula, and sampled exactly as indicated above for the preterm pigs. All of the studies were approved by the National Committee on Animal Experimentation, Denmark.
Intestinal Proteome Analysis
Intestinal proteomic analysis was performed in 2 subgroups of 1-day old, formula-fed preterm pigs (n = 5, cesarean delivered at 92% gestation) and term pigs (n = 5, spontaneously delivered at full term). The gel-based proteomics did not allow for high-throughput analysis of all pigs from the experiment described above. The chosen subgroups of pigs were representative of the remaining pigs in their groups with regard to average body weight and intestinal characteristics. None of the term pigs showed any sign of abnormality at tissue collection, whereas the preterm pigs showed mild degrees of macroscopic lesions in the mid-intestinal segment that was subjected to proteome analyses. The median NEC lesion score for the investigated mid-intestinal section was 3 for preterm pigs and 1 for the term pigs (nonparametric Mann-Whitney U test, P < 0.05).
Tissue protein extraction, 2-dimensional gel electrophoresis (2-DE), and mass spectrometry (MS) for protein identification were carried out as described previously (7,8). Briefly, the mid-intestine tissue samples from preterm and term pigs were disrupted in a cocktail buffer (1% Triton X-100, 25 mmol/L Hepes, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid disodium salt, 1 mmol/L dithiothreitol) containing protease inhibitor. The protein extract was obtained by centrifuging at 15,800g for 30 minutes at 4°C; the superfluous salt was removed by trichloroacetic acid-acetone precipitation (7,12). Protein extracted was resuspended in a buffer containing 7 mol/L urea, 2 mol/L thiourea, and 4% Chaps. Protein solution was stored at −80°C until being used for 2-DE. Protein concentration of the samples was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).
One gel was run for each intestine from both groups. The protein sample (100 μg) was mixed with 350 μL rehydration buffer (9.5 mol/L urea, 2% Chaps, 0.28% dithiothreitol, 0.5% immobilized pH gradient (IPG) buffer pI 3–10), transferred onto one 18-cm ReadyStrip IPG Strips (pI 3–10 nonlinear, Bio-Rad). The isoelectric focusing was carried out on an Ettan IPGphor III IEF System (GE Healthcare, Uppsala, Sweden) after an active rehydration step following a 5-step program. The proteins were further separated by the 2-DE using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with focused gel strips placed on top of 1.0-mm thick 12.5% polyacrylamide gels. After electrophoresis, gels were stained with SYPRO Ruby Protein Stain (Bio-Rad) according to the manufacturer's guide. Stained gels were scanned with the Molecular Imager PharosFX Plus System (Bio-Rad) and analyzed by 2-DE analysis software PDQuest 8.0 (Bio-Rad). Spots that matched among the gels were assigned with numbers. The expression level of matched spots was exported to SPSS 11.5 for statistical analysis (SPSS Inc, Chicago, IL).
Spots with significant difference in expression abundance were picked out from the gels and digested with trypsin (7,8). The tryptic digests were subjected to protein identification with MS on a 4800 MALDI TOF/TOF MS Analyzer (Applied Biosystems, Carlsbad, CA) with taxonomy as Mammalia for protein identity searching.
A 30-μg protein of every mid-intestine extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies used were anti-guanine nucleotide-binding protein-2-like 1 (Gnb2l1) (Abcam, Cambridge, UK), anti-prohibitin (Abcam), anti-β-actin (Sigma-Aldrich, St Louis, MO), and anti-arginosuccinate synthetase (ASS) (BD Biosciences, Franklin Lakes, NJ) to assess the expression of Gnb2l1, prohibitin, β-actin, and ASS. The protein bands were visualized and analyzed with the band density–analyzing software Quantity One (Bio-Rad).
Multivariate and Univariate Statistical Analyses
A principal component analysis (PCA) of the proteomic expression data was carried out using LanteniX (Latent5, Copenhagen, Denmark). Nonparametric Mann-Whitney U test of the NEC lesion scores was done with GraphPad Prism (GraphPad Software, La Jolla, CA). Two-tailed Student t test using SPSS 11.5 was used to compare the difference in the means of organ dimensions, brush border enzyme activities, 2-DE protein expression quantity, and band intensity of Western blot. All of the statistical analyses with P value <0.05 were considered statistically significant.
Preterm and Term Pigs at Birth and After 1 Day of Feeding
The organ weights and brush border enzyme activities for newborn and 1-day-old preterm and term pigs are shown in Figures 1 and 2. All of the pigs had similar body weight at birth and 1 day later, although there were trends for preterm pigs to lose weight and for term pigs to gain weight. Preterm pigs had significantly lower body weight than term pigs after feeding (1.05 ± 0.01 vs 1.37 ± 0.08, mean ± standard error of the mean, P < 0.05, Fig. 1A). Intestinal relative weight was also similar between delivery groups at birth and after 1 day of feeding, and both groups showed a massive approximately 60% increase in intestinal-to-body weight ratio (P < 0.05, Fig. 1B), associated with large increases in the proportion of mucosa (P < 0.05, Fig. 1D). Preterm pigs showed increase in intestinal length after feeding, although less than in term pigs (296 ± 17 vs 393 ± 15 cm, mean ± standard error of the mean, P < 0.05, Fig. 1C). Preterm pigs did not show an increase in villous height in response to feeding (Fig. 1E), probably because of variable degrees of intestinal atrophy. Across the 36 preterm pigs fed formula, 12 showed macroscopic signs of NEC in the small intestine, whereas none of the term pigs had NEC lesions (33% vs 0%, P < 0.05). NEC incidence and severity values were similar to those in our previous studies using an identical protocol (4). Regardless, the feeding-induced decrease in lactase activity (Fig. 2A) and increase in maltase activity (Fig. 2B) in the middle small intestine were extremely similar between preterm and term pigs. Sucrase activity responded to feeding, with a decrease only in preterm pigs (Fig. 2C). None of the 3 peptidases showed significant difference between unfed and fed preterm or term pigs, except for a tendency for preterm pigs to show increased activity of aminopeptidases N and A after feeding (P < 0.10, Fig. 2D, F).
Intestinal Proteome Analysis
Five gels from each group were applied to expression analysis by PDQuest. Approximately 500 spots were detected on each gel (Fig. 3). Seventy-one spots on the 2-DE gels showed >2-fold expression difference between the groups with statistical significance (P < 0.05). In total, 36 proteins were identified successfully by MALDI-TOF/TOF MS, with protein score >71 (Fig. 3). Detailed information of the identified proteins, such as spot number, protein name, Geninfo identifier (GI Id), protein score, expression level in preterm and term piglets, and the ratio of expression level in preterm group over term group, is listed in Table 1. A score plot of PCA analysis of intestinal proteome of both preterm and term pigs is shown in Figure 4. The first 2 components of this model accounted for approximately 54% of variation in the data. Preterm pigs showed a more scattered pattern than term pigs, suggesting that these pigs had a less homogeneous intestinal proteome on day 1 of life.
Among the 36 identified proteins, 10 proteins (28%) showed elevated expression in preterm pigs compared with term pigs, whereas 26 of them (72%) showed higher expression in the term group. The major biological functions of the identified proteins relate to stress response and detoxification, protein processing, intracellular trafficking, signal transduction, metabolisms of fatty acid, carbohydrate, arginine and energy, cytoskeleton and cell mobility, and actions of the enteric nervous system.
We cross-validated the 2-DE results of 4 selected proteins by Western blots. Prohibitin, β-actin, and ASS showed significant reduction (P < 0.05), whereas Gnb2l1 tended to be reduced in the preterm versus term pigs (Fig. 5).
It remains unknown how immaturity contributes to the elevated NEC sensitivity of the intestine in preterm neonates. In the present study in newborn pigs, the immediate structural and functional responses of the premature intestine to enteral feeding were similar to that in pigs born close to term, whereas preterm pigs showed a higher incidence of macroscopic NEC-like lesions and many differences in intestinal protein expressions, potent resulting from the state of physiological immaturity as well as from the formula-induced proinflammatory condition of the intestine. The PCA analysis of the proteomic result showed higher variation among the preterm pigs, and the intestinal proteome of the preterm pig with the lowest NEC score (pig 106, NEC score 1) appeared closest to the cluster of proteomes from term pigs in the PCA score plot (Fig. 4). Differences in delivery method are not likely to contribute to this variation between the preterm and term pigs because delivery method does not alter the feeding-induced changes in intestinal functions or NEC sensitivity in preterm pigs (6). We have consistently observed that formula feeding, combined with immaturity, tends to overshadow any influence of factors such as delivery mode, birth colonization, and probiotics on the immature intestine (13). Here we show that this dramatic effect of enteral feeding with formula just after birth occurs only in preterm neonates and is associated with a series of changes (mainly reductions) in expression of intestinal proteins.
Among the identified proteins, 10 proteins showed elevated expressional level in preterm pigs and they were complement component 3 (C3), glutathione S-transferase π (GST π), 3 gelsolin precursors, keratin 9, dihydropyrimidinase-related protein 2 (DPYSL2), doublecortin domain-containing protein 2 isoform 1 (DCDC2), serotransferrin, and albumin. C3 is a component of the complement system, a part of the innate immune system. With undeveloped adaptive immune systems, the neonates mainly rely on the innate immune system to fight pathogens (14). The action of C3 is correlated with Toll-like receptor 4 and with intestinal ischemia-reperfusion (15), both of which play key roles in NEC in infants and animal models (16) and may relate to gut microbe density as shown in our study on oral antibiotics in preterm pigs (10). DPYSL2 and DCDC2 are associated with the development of enteric nervous system (ENS) with DPYSL2 being involved in axonal outgrowth and path finding (17), suggesting differences existed in the ENS between the preterm and term pigs. Previous studies have related NEC with dysfunctional enteric glial cells (18), and our own studies in preterm pigs suggest that the effects on ENS may be highly age and diet dependent (19,20). Both the tissue complement system and the ENS are overlooked in NEC etiology, and our proteomic analysis shows that these systems warrant further investigations as therapeutic targets against prematurity-related intestinal dysfunction.
During the process of feeding-induced NEC in premature neonates, an oxidative stress response may be related to the action of pathogenic microbes and their toxins. This colonization-dependent intestinal stress response was already evident from our previous proteomic studies on control and gnotobiotic fetal and neonatal preterm pigs (7,12). GST π, a part of the glutathione/GST antioxidant system, protects cells against oxidative stress and epithelial damage (21,22). The expression of prohibitin is related to that of GST π, and it protects from oxidative depletion of glutathione (22). Prohibitin was downregulated in the preterm pigs as in active Crohn disease and experimental colitis (22). The level of prohibitin may initially decrease in response to cellular stress in preterm neonates; however, with prolonged feeding and NEC lesions, the protein levels increase (8). Modulating prohibitin level may serve as a new therapeutic strategy for treating NEC (22). In the present study, the decreased expression of both prohibitin and GST π in preterm pigs may suggest that the newborn immature intestine indeed has defects in its antioxidative and stress-response capacity.
Three proteins related to protein processing (proteasome subunit β4, heat-shock cognate 70-kDa protein, and matricin) and 2 proteins associated with intracellular trafficking between nucleus and cytoplasm or with Golgi apparatus (vacuolar protein sorting 29, heterogeneous nuclear ribonucleoprotein A2/B1 isoform 2) had decreased expression in preterm pigs. Proteasome subunit β4 is a main regulator of protein degradation and removal of damaged and short-lived regulatory proteins (23), whereas matricin stimulates folding and oligomeric assembly of newly synthesized proteins (24). Heat-shock cognate 70-kDa protein binds to nascent polypeptides to facilitate correct protein folding (25). Heterogeneous nuclear ribonucleoproteins processes degrades RNA and transfer RNA between nucleus and cytoplasm transport (26,27), whereas vacuolar protein sorting 29 transfers proteins (28). Because these proteins were not affected by feeding or bacterial colonization in the previous studies on preterm pigs (7,9), we conclude that prematurity is associated with a less active intestinal protein metabolism.
Arginine metabolism is particularly important in NEC progression because it relates closely to nitric oxide production, which is important in maintaining mucosal integrity and protection from blood-born toxins and tissue-destructive mediators (29). Two proteins related to arginine metabolism (ornithine carbamoyltransferase and ASS) had lowered expression in preterm pigs suggesting an impaired intestinal arginine metabolism. Ornithine carbamoyltransferase is involved in formation of citrulline, whereas ASS is a rate-limiting enzyme in arginine synthesis.
In our previous studies, decreased expression pattern of proteins associated with metabolism of carbohydrate, fatty acid, and energy was found in preterm pigs in response to feeding and/or bacterial colonization (7–9). We conclude that the immature intestine is in a relatively energy-deficient physiological state after the first enteral feeding, and this may enhance its sensitivity to inflammatory lesions. Similarly, 5 proteins associated with carbohydrate, 1 with fatty acid, and 5 with energy metabolism were identified with decreased expression in preterm versus term pigs in the present study (Table 1). Among the identified proteins, succinate dehydrogenase is involved in oxidative phosphorylation (30) and electron transfer flavoproteinubiquinone oxidoreductase is involved in electron transfer in the mitochondrial respiratory chain (31), whereas fumarate hydratase functions in the citric acid cycle. Λ-crystallin was repeatedly found with decreased expression in our previous studies with lower expression in preterm pigs with NEC (9,10,12). The decreased lactate dehydrogenase results in decreased catalysis of the reversible NAD-dependent interconversion of pyruvate and lactate (32). Phosphoglycerate mutase 1 and phosphoglycerate kinase 1 are involved in the glycolysis process (33), and phosphoglycerate kinase 1 is also a target of hypoxia-inducible factor 1α (HIF1α) (34). HIF1α regulates transcription of genes that encode protein for tissue oxygen delivery under conditions of impaired oxygen availability, such as inflammatory conditions (35).
Another identified protein, Gnb2l1, also reacts with HIF1α. It competes with heat shock protein 90 for binding HIF1α and promotes its degradation (35). Hence, the reduction in Gnb2l1 in preterm pigs may contribute to impaired oxygen delivery and energy metabolism. In fact, intestinal Gnb2l1 protein was significantly upregulated in response to formula feeding of fetal pigs in utero (12), probably reflecting that the pronounced feeding-induced trophic responses in fetuses are not associated with compromised regulation of tissue metabolism, like in the preterm neonates.
Both up- and downregulation of proteins related to cytoskeleton and cell mobility were found in the present study. Gelsolin (upregulated in preterm pigs) has not previously been related to NEC, but its major function in rearranging actin cytoskeleton by capping and severing F-actin (36) suggests that it may have a role in the reaction between actin and microbial attack. Finding of three isoforms of gelsolin also suggests that this protein could be posttranslationally modified in certain ways. Some other proteins involved in formation and rearrangement of actin cytoskeleton, chloride intracellular channel protein 1 (37) and WD repeat1 protein (38), and an actin component showed lower expression in the preterm pigs.
Compared with term pigs, preterm pigs fed formula for 1 day showed similar trends in intestinal maturation and growth despite that they had markedly different expression of proteins involved in multiple cellular processes, ranging from tissue stress response to metabolism. Complement 3, prohibitin, arginine metabolism proteins (ornithine carbamoyltransferase and ASS), and ENS proteins (DPYSL2 and DCDC2) are good targets for further studies and may possess the potential to serve as prognostic markers or therapeutic targets. Dietary or medical interventions that manipulate these proteins, or the processes they are involved in, may decrease the risk of NEC when enteral feeding is initiated after preterm birth.
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Keywords:Copyright 2013 by ESPGHAN and NASPGHAN
enteral formula feeding; necrotizing enterocolitis; preterm neonate; proteomics; term neonate