Introduction

Low birth weight in piglets correlates with lower survival rates, permanent negative growth performance and sub-optimal carcass quality [1, 2] thus representing a serious agronomical problem. Remarkably, genetic selection has resulted in an increase proportion of piglets with low birth weight over the last decade. Piglets with a birth weight close to the mean body weight (BW) (±0.5 SD) are identified as normal BW animals while piglets with a mean −2 SD lower BW (−30 %) are considered as piglets with intrauterine growth retardation (IUGR) [3]. IUGR is known to reduce the thickness and weight of stomach, small intestine and colon, as well as the density and length of small intestine and colon, which result in a reduction in the intestinal surface area for absorption [4–6]. In addition, the intestinal functions have been reported to be impaired in piglets with low BW at birth with lower lactase and aminopeptidase N activities in intestine, as well as lower relative weight of pancreas [6]. Intestinal mucosal growth is known to be very intense after birth in normal BW piglets [7].

Glutamate is one of the major oxidative fuel for the intestine, but also serves as an important precursor for other biologically active molecules, such as glutathione, arginine, proline, alanine, aspartate, ornithine, citrulline and N-acetylglutamate [8–11]. Energy metabolism is particularly high in enterocytes. Indeed, although the intestine represents only about 5 % of BW, it utilizes about 20 % of whole-body oxygen consumption [12]. It is widely accepted that gastrointestinal tissues derive their oxidative energy mainly from the catabolism of amino acids (AA), rather than from glucose or fatty acids oxidation [13]. The main AA catabolized in the gut are glutamate, glutamine and aspartate [14]. Mucosal glutathione is mainly metabolized from enteral l-glutamate in fed piglets and inhibition of mucosal glutathione synthesis is associated with intestinal dysfunction which can be restored by giving glutathione monoester orally [15].

The first step of glutamate metabolism is transport across the apical brush border membrane from the gastrointestinal lumen. The high-affinity X −AG system involved in transport of both l-glutamate and l-aspartate is the most important transport system of l-glutamate. Excitatory amino acid carrier 1 (EAAC1), as the most abundant member of the X −AG system, is mainly expressed in the small intestine (especially jejunum) [16]. EAAC1 was found to be expressed in the brush-border membrane of isolated epithelial cells from both villus and crypt origin [17, 18]. The efficiency and capacity of transport of luminal L-glutamate across the apical membrane are regulated by changes in the expression of EAAC1 [17]. In that overall context, the present study was conducted with suckling Huanjiang mini-pig as animal model to test the hypothesis that marked differences in jejunal EAAC1 expression profiles in piglets can be associated with large body weight (LBW) or small body weight (SBW). Previous work from our laboratories have shown that piglet with low BW at birth are characterized by low expression of the B0AT1, ASCT2 and b°,+ transporter in the jejunum [19, 20]. The objective of the present work was to clone the sequences of EAAC1 from Huanjiang mini-pig, and to measure the expression profile of the jejunal EAAC1 in the newborn and suckling piglets with LBW or SBW.

Materials and methods

Animals and tissue sample collection

Twenty litters of Huangjiang mini-pigs were spontaneously delivered from sows at term (~114 days of gestation). At birth, two piglets (one with the largest BW and another with the smallest BW) were selected from each of the 20 litters and weighed immediately. On day 0, five LBW and five SBW piglets were pairs from the same five litters, and these piglets were not allowed to suckle milk from the sows up to euthanasia. The rest of the selected piglets (15 LBW and 15 SBW) were positioned in the second teat pairs for suckling from their own mother and used at the different time points that are days 7, 14 and 21. Suckling piglets were helped for fixing nipple to prevent fight during suckling. Piglets then get used to fix a given nipple during the suckling period. These suckling piglets were weighted 1 h after the last suckling (see Table 1), and anaesthetized prior to tissue recovery and euthanasia. General anaesthesia was obtained via intravenous injection of 4 % sodium pentobarbital solution (40 mg/kg BW) and killed by jugular puncture [21]. Samples of anterior jejunum (5 cm, after cleaning by ice-cold phosphate-buffered saline) were collected and immediately frozen in liquid nitrogen and stored at −70 °C until analysis. This study was conducted according to the guidelines of the Declaration of Helsinki and all procedures involving animal subjects were approved by the animal welfare committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences [22].

Table 1 Body weight as a function of age for piglets with LBW and SBW
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Cloning of the EAAC1 cDNA

Total RNA was extracted from jejunum using TRIZOL Reagent (Invitrogen, CA, USA). The integrity of the RNA was verified by electrophoresis in 1 % agarose gel stained with ethidium bromide. The quality and quantity of RNA were determined by ultraviolet spectroscopy using a NanoDrop® ND-1000 (Thermo Fisher Scientific, DE, USA). Total RNA was reverse-transcribed using the random primer Oligo-dT18 and Moloney Murine Leukemia Virus reverse transcriptase (Promega, WI, USA). Primers used to recognize the EAAC1 cDNA sequence of Huanjiang mini-pig were designed using Oligo 6.0 software based on the human and mouse EAAC1 cDNA sequences. The full-length cDNA of EAAC1 was amplified from the jejunum cDNA by RT-PCR using EAAC1 primers [1] (Table 2) and 2× Taq PCR Master Mix (Tiangen Biotech, Beijing, China). The PCR program consisted of 30 cycles of 94 °C for 30 s, 52 °C for 45 s, and 72 °C for 2 min. The PCR products were separated by electrophoresis on 2 % agarose. The purified PCR product was cloned into the pGEM-T easy Vector (Promega, USA) and sequenced at Beijing Genomics Institute [23, 24].

Table 2 Primers used for cloning and real-time PCR analyzing
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Relative quantification of EAAC1 gene expression

Primers for selected genes (Table 2) were designed using Oligo 6.0 software. Real-time quantitative PCR analyses were performed with 5 ng of reverse-transcribed RNA and both sense and anti-sense primers in a final volume of 10 μl using SYBR Green I as a PCR core reagent. After a pre-denaturation program (10 s at 95 °C), forty cycles of amplification were conducted with each cycle consisting of 95 °C for 10 s, 60 °C for 20 s, and following by a melting curve program (60–99 °C with heating rate of 0.1 °C/s and fluorescence measurement). The amplification of GAPDH was used for each sample to normalize the expression of the selected genes.

Determination of EAAC1 protein quantity

Frozen samples were powdered under liquid nitrogen, and lysed in RIPA buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris–HCl at pH 7.4, plus a protease inhibitor cocktail purchased from Roche, Shanghai, China). After centrifugation at 10,000×g and 4 °C for 10 min, the protein concentration in the supernatant fluid was determined using the Bicinchoninic Acid assay (Beyotime biotechnology, China). All samples were adjusted to an equal protein concentration and then diluted with 2× loading buffer (0.63 ml of 0.5 M Tris–HCl (pH 6.8), 0.42 ml 75 % glycerol, 0.125 g sodium dodecyl sulfate (SDS), 0.25 ml β-mercaptoethanol, 0.2 ml 0.05 % solution of bromphenol blue, and 1 ml water) to a final volume of 2.5 ml and heated in boiling water for 5 min. After boiling, the samples were cooled on ice and used for Western blot analysis.

The denatured proteins (25 μg per sample) were separated using SDS-PAGE (4–12 % gradient gel), transferred to PVDF membranes (Millipore, Billerica, MA) overnight at 12 V using the Bio-Rad Transblot apparatus (Hercules, CA). The membranes were blocked in 5 % fat-free milk in Tris-Tween buffered saline (TTBS; 20 mM Tris/150 mM NaCl, pH 7.5, and 0.1 % Tween-20) for 3 h and then incubated with EAAC1 or β-actin antibody (Santa Cruz, CA, USA) at 4 °C overnight with gentle rocking. After washing three times with TTBS, the membranes were incubated at room temperature for 3 h with horseradish peroxidase-linked secondary antibodies (Santa Cruz, CA, USA). The primary antibody and secondary antibody were used at dilutions of 1:1,000 and 1:3,000, respectively. Finally, the membranes were washed with TTBS, followed by development using Supersignal West Dura Extended Duration Substrate according to the manufacturer’s instructions (Pierce, Rockford, IL). The images were detected on chemiluminescence (Applygen Technologies Inc., Beijing, China). Multiple exposures of each Western blot were performed to ensure linearity of chemiluminescence signals. Western blots were quantified by measuring the intensity of correctly sized bands using AlphaImager 2200 (Alpha Innotech Corporation, CA, USA) software.

Bioinformatics analysis

Sequences were aligned with the multiple alignment program Clustal W. The putative extra-cellular N-glycosylation sites, deduced protein kinase C phosphorylation sites and potential cAMP/cGMP-dependent protein kinase phosphorylation sites were analyzed in silico by PIR (http://pir.georgetown.edu/pirwww/search/pattern.shtml). The trans-membrane helices were predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The AA sequences were analyzed using MotifScan (http://myhits.isb-sib.ch/cgi-bin/motifscan). The putative signal peptides were predicted by Signal P 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) and the putative cut sites of signal peptides predicted by ProP 1.0 Server (http://www.cbs.dtu.dk/services/ProP/). Phyre (http://www.sbg.bio.ic.ac.uk/~phyre/index.cgi) was used to predict the tertiary structure. The hydrophobicity of deduced protein was analyzed by Protscale (http://www.expasy.ch/tools/protscale.html). MEGA 5 was used for phylogenetic analysis.

Statistical analysis

All data were subjected to paired t test using the SAS version 9.2 Programme (SAS Institute, Cary, NC). Data are presented as mean ± SEM and differences with P values <0.05 were considered as being statistically significant.

Results

Identification and characterization of EAAC1 cDNA in Huanjiang mini-pig

The ORF of EAAC1 cDNA in Huanjiang mini-pig was 1,575 bp in length encoding a 524-AA polypeptide (Fig. 1). The cDNA sequence shared 99.30 and 99.43 % identity with Tibet pig and common pig, and shared 89.97 and 84.13 % with human and mouse, respectively. Three putative extra-cellular N-glycosylation sites and one deduced cAMP/cGMP-dependent protein kinase phosphorylation site were identified (Fig. 1). Analysis of the AA sequence suggested the presence of a N-terminus signal peptide (Fig. 2), which was confirmed by the putative cut site (between Glu31 and Ile32) predicted by ProP 1.0 Server. Eight proposed trans-membrane domains were identified (Fig. 2), in good agreement with the hydrophobicity analysis (Fig. 3a). Two proposed “Sodium: dicarboxylate symporter family signature” were found to be highly conserved when compared with other animal models (Fig. 2). The predicted three dimensional structure was constructed (Fig. 3b) and the phylogenetic analysis of the AA sequence was performed. As shown on Fig. 4, the neighbor-joining tree showed that the Huanjiang mini-pig had a closer genetic relationship with cow when compared with the other animals examined.

Fig. 1
figure 1

Nucleotide and deduced amino acid sequences of excitatory amino acid carrier 1 in Huanjiang mini-pig. The stop codon is indicated by asterisk. Putative extracellular N-glycosylation sites (shed in grey) and potential cAMP/cGMP-dependent protein kinase phosphorylation site (highlighted in box) were predicted by PIR

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Fig. 2
figure 2

Amino acid sequence alignment of excitatory amino acid carrier 1 (EAAC1). Sequences from Huanjiang mini-pig (pEAAC1), cow (cEAAC1), human (hEAAC1), mouse (mEAAC1), and rat (rEAAC1) were aligned by Clustal W. The putative trans-membrane segments (TM1–TM8) of Huanjiang mini-pig were predicted using the TMHMM 2.0 program. The proposed “Sodium: dicarboxylate symporter family signature” were predicted by MotifScan and PIR and shed in grey. Putative signal peptides highlighted in box were predicted using SignalP 3.0 Server

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Fig. 3
figure 3

Hydrophobicity and three dimensional structure prediction for excitatory amino acid carrier 1 (EAAC1) in Huanjiang mini-pig. a Hydrophobicity oscillograms of EAAC1 was depicted using Protscale with the option Hphob./Eisenberg et al. b The predicted three dimensional structure was constructed by Phyre based on template-based homology modeling

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Fig. 4
figure 4

Phylogenetic analysis of excitatory amino acid carrier 1 in Huanjiang mini-pig. The unrooted tree was constructed using neighbor-joining (NG) method based on the alignment of the complete amino acid sequences by MEGA 5.0. Bootstrap values at each node represent the percentage of 100 replicates

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Body weights of LBW and SBW piglets

As indicated on Table 1, the procedure of piglet selection, according to their BWs at birth, allowed to show that the initial reduction of the BW in the SBW group (i.e. 56 % of the mean BW in the LBW group) was maintained during all the suckling period averaging 55 % at day 7, 55 % at day 14, and 50 % at day 21.,

Expression level and amount of jejunal EAAC1 in suckling Huanjiang mini-piglets with LBW or SBW

As indicated in Fig. 5, the measurement in the jejunum of the expression profile of EAAC1 mRNA in LBW piglets revealed that there was a modest increased expression of the carrier between birth and after 7 days of suckling. In contrast, a spectacular increase of EAAC1 expression (more that fivefold increase) was recorded between days 7 and 14 of the suckling period. Then, the expression of the carrier tended to decrease between days 14 and 21. The pattern of expression was found to be similar in SBW animals. However, in the SBW animals, the initial low expression of the EAAC1 carrier was maintained during the suckling period up to day 14. The measurement of the expression of EAAC1 as protein in piglet jejunum revealed that in LBW the protein expression follows the mRNA expression with protein expression being similar between birth and after 7 days of suckling; but major increase between days 7 and 14 animals (Fig. 6). In SBW piglets, the pattern of EAAC1 protein expression was similar when compared with LBW animals, with the carrier being much less expressed in SBW animals both at birth and after 7 days sucking.

Fig. 5
figure 5

mRNA abundance of jejunal excitatory amino acid carrier 1 (EAAC1) in suckling Huanjiang mini-piglets with LBW and SBW from days 0 to 21 of age. Samples were obtained at days 0, 7, 14 and 21 of age, respectively. The mRNA expression levels of EAAC1 were normalized using GAPDH as an internal control. Values with asterisk on bars differ (P < 0.05) when compared with the SBW piglets. Data are expressed as mean ± SEM, n = 5. LBW large body weight, SBW small body weight

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Fig. 6
figure 6

Protein abundance of jejunal excitatory amino acid carrier 1 (EAAC1) in suckling Huanjiang mini-piglets with LBW and SBW from days 0 to 21 of age. Jejunum samples were obtained at days 0, 7, 14, and 21 of age, respectively. The protein expression levels of EAAC1 were normalized using GAPDH as an inner control. Values with asterisk on bars differ (P < 0.05) when compared with the SBW piglets. Data are expressed as mean ± SEM, n = 5. LBW large body weight, SBW small body weight

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Discussion

The mini-pig models are catching an increasing attention from the community of physiologists because its anatomical, physiological and metabolic characteristics are more similar to human than other animal models like rodents [25]. In addition, this animal is convenient to manipulate and less expensive compared with large domestic animals, because of its small size [26]. Thus, the mini-pig is a timely model for research on intestinal physiology and development. Huanjiang mini-pig is a Chinese indigenous breed of pig, which is mainly distributed in Huanjiang county, Guangxi province [27]. It has thus been widely used for basic research [28–30] and attracted strong agronomic interests due to the high quality carcass, which is relatively rich in proteins and low in fat [25, 31].

In order to measure the expression profiles of intestine EAAC1 in pigs with different BW during suckling period, we first cloned the sequence of EAAC1 from Huanjiang mini-pig by PCR method. Molecular cloning found that the ORF of EAAC1 cDNA sequence in Huanjiang mini-pig was 1,575 bp, which encoded a deduced protein with 524 AA. The deduced protein has the same size with human [32] and cow [33], and one more AA than mouse [34] and rat [35]. The EAAC1 carrier of Huanjiang mini-pig belongs to the sodium: dicarboxylate symporter family and has two sodium: dicarboxylate symporter family signatures, an information that was predicted by MotifScan and confirmed by PIR programs [36, 37]. The structure of the member in this family usually has 8–10 trans-membrane regions. In accord with these characteristics, the EAAC1 of Huanjiang mini-pig was found to possess eight putative trans-membrane domains with the same number of mouse and rat, and one more than human and cow as predicted by TMHMM 2.0 program [38]. The function of glutamate transporter can be regulated by phosphorylation and glycosylation [39], and all of its members share two evolutionarily conserved glycosylation sites with some members having one more or one less site [35]. Three potential N-linked glycosylation sites were identified, including two conserved sites. Protein kinase C and cAMP/cGMP-dependent protein kinase are known to be involved in regulating the activity of glutamate transporter [40, 41], and potential phosphorylation sites were identified in EAAC1. Meanwhile, the phylogenetic tree showing that Huanjiang mini-pig had a closer relationship with human than mouse and rat, indicates that Huanjiang mini-pig is likely a better model than mouse and rat for the study of the function of EAAC1.

Immediately after birth, the suckling period is a very critical time for mammals, with important consequences for the growth and further development of cells, tissues and organs including particularly the intestines. The intestine of suckling neonates with low BW shows different pattern of maturation when compared with the neonates with normal BW [6]. It is quite clear that the intestine of neonates with low BW is associated with important changes of the morphology, physiology and metabolism [5, 42], but the concomitant changes at the molecular level remain unclear. The present study showed that, compared with the LBW pigs, Huanjiang mini-pigs with SBW expressed a lower level of EAAC1 mRNA and protein in jejunum after the first week of birth, such period being considered as critical for further development. Considering that EAAC1 is the key transporter for glutamate [17], and that glutamate, apart from its function of a building block for protein synthesis and as precursor of AA and glutathione de novo biosynthesis; is one of the most important fuel for pig enterocytes [43, 44]; the abnormal expression of EAAC1 in SBW piglets may be associated in a vicious cycle with abnormal intestinal growth development and abnormal absorptive functions. Further work which is out of the scope of the present study should be conducted to test whether the reduction of the expression of EAAC1 in the jejunum of SBW piglets play a major role and is causally linked with delay in the development of the SBW piglets. In addition, in order to fully evaluate the consequence of presumed limited glutamate supplying to enterocytes, one should evaluate the supplying of other energy substrate (e.g. glutamine) to the intestinal epithelial cells. Regarding this latter point, it is worth to consider that, at difference with glutamate which is transaminated in the presence of oxaloacetate and pyruvate to alpha-ketoglutarate (and respectively aspartate and alanine) before entry of alpha-ketoglutarate into mitochondria for utilization in the TCA cycle; glutamine must firstly enter the mitochondria and be degraded to ammonia and glutamate by the phosphate-dependent glutaminase before export of glutamate into the cytosol where this amino acid is converted to alpha-ketoglutarate before finally reentering the mitochondria and the TCA cycle [8]. In other words, the utilization of glutamine as energy substrate in enterocytes implies more metabolic steps than the utilization of glutamate for ATP production.

From an agronomical perspective, the piglets with low birth weight are often eliminated for their increased susceptibility to the onset of organ dysfunction, permanent impaired growth and sub-optimal carcass quality [2, 45] representing an important commercial concern. We propose that the low birth weight of piglets may thus restrict the intestinal capacity to transport glutamate and this might further limit the development and absorptive functions of the intestine and thus play a role in the delayed growth of piglets with an already lower birth weight. More research is needed in order to test new strategies aiming at improving expression of EAAC1 in piglets born with a SBW and testing it for its capacity to ameliorate intestinal absortive functions and growth.