Comparative Biochemistry and Physiology, Part B 164 (2013) 194200

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Comparative Biochemistry and Physiology, Part B

journal homepage: www.elsevier.com/locate/cbpb

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

Zhen Liu, Yi Zhou, Junchang Feng, Shuangqing Lu, Qiong Zhao, Jianshe Zhang
Department of Biotechnology and Environmental Science, Changsha University, Changsha 410003, China

a r t i c l e

i n f o

a b s t r a c t
The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression prole in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether sh diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a signicant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with sh meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the rst time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a signicant effects on PepT1 gene expression in grass carp. 2013 Elsevier Inc. All rights reserved.

Article history: Received 7 September 2012 Received in revised form 19 November 2012 Accepted 26 November 2012 Available online 4 December 2012 Keywords: Oligopeptide transporter Grass carp Dietary protein Sodium butyrate SLC15A1

1. Introduction Teleosts, unlike other vertebrates, require high protein diets to obtain amino acids for protein synthesis and energy metabolism. In teleosts, dietary proteins are eventually degraded into a mixture of free amino acids and small peptides in the intestine. A large number of studies have shown that protein digestion products are mainly present in the form of small peptides such as di- and tri-peptides that are absorbed by the di- and tri-peptides transporters in intestine (Gatlin et al., 2007). Thus, a better understanding of the regulation of protein absorption by the intestinal transporter is important for improving dietary nutrition, and has a potential application in aquaculture and sh breeding. The cell absorption of di- and tri-peptides is mediated by members of the so-called solute carrier (SLC) family. PepT1 (or SLC15A1), as a member of this family, is responsible for transporting of small peptides across the bush-border membrane of the small intestinal epithelium (Daniel, 2004). Several studies have shown that PepT1 plays a critical role in small peptides transportation using heterologous expression systems. In addition, studies on regulation of PepT1 expression have
Corresponding author. Tel./fax: +86 731 84261452. E-mail address: jzhang@ccsu.cn (J. Zhang). 1096-4959/$ see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpb.2012.11.008

shown that intestinal PepT1 is regulated by a variety of hormones (Terada and Ki, 2004), epidermal growth factor (Nielsen et al., 2001), diurnal rhythm (Pan et al., 2004) and dietary protein content (Shiraga et al., 1999). As demonstrated by Erickson et al. (1995), PepT1 mRNA expression and peptide transport rate in rat intestine could be stimulated by protein diet. The PepT1 cDNAs have been identied and cloned in shes, including zebrash (Danio rerio) (Verri et al., 2003), Atlantic cod (Gadus morhua) (Rnnestad et al., 2007) and Common carp (Cyprinus carpio) (Ostaszewska et al., 2009). These sequences give a good base for evaluation of PepT1 gene expression in these species. As demonstrated by Dring et al. (1998), PepT1 was highly expressed in the small intestine, but expression levels were lower in the kidney and bile duct epithelium. Several studies have shown that PepT1 expression was temporally and spatially regulated in sh embryos and adult zebrash, which reected the prole of small peptide transport function (Verri et al., 2003; Romano et al., 2006). Moreover, it has been reported that PepT1 expression was also modulated by diets in teleosts (Gonalves et al., 2007; Amberg et al., 2008; Hakim et al., 2009; Terova et al., 2009; Ostaszewska et al., 2010). As a representative of the freshwater species from the Cyprinidae, the grass carp (Ctenopharyngodon idella) is a native Chinese freshwater sh with a broad distribution from the catchment area of the Pearl River

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in the southern China to the Heilongjiang River in northern China. The grass carp has been introduced to over 40 other countries, and is the most economically relevant cultured freshwater sh in many regions of the world. The aquaculture of grass carp relies on sh feed. Fish meal has been traditionally used as the main protein source in the preparation of diet for cultured grass carp. However, a limited world supply combined with the price increase of sh meal has recently led to the replacement of sh meal with alternative protein sources, mainly of vegetable origin (Conceio et al., 2012). However, the new formulations have resulted in enteritis and low growth rate in some aquatic species such as Atlantic salmon (Salmo salar) and other carnivorous sh (Verri et al., 2010). Thus, studies of small peptide transport offered a window of opportunity to better understand the mechanisms in this process and potential improvement of sh diet with different formulations. Because PepT1 is a key transporter of di/tripeptides, PepT1 has been recently considered as an important target for formulating diets. The studies on PepT1 would help researchers to design novel diets with more appropriate protein compositions for better absorption. The aim of this study was to clone the PepT1 cDNA from grass carp and studied its expression in different tissues of the grass carp. In addition, we characterized PepT1 expression in the early development and diurnal rhythm by realtime RT-PCR. Moreover, we extended our studies to determine the inuence of dietary protein and feed additives on PepT1 expression. These studies provide valuable knowledge on the regulation of PepT1 expression in sh and the effect of sh diet on PepT1 expression, and thus have a potential application in improving sh feed formulation in aquaculture. 2. Materials and methods 2.1. Animals and tissue preparation Grass carp were provided by the Hunan Institute of Aquatic Science. After animals were anesthetized by 2-phenoxyethanol (Sigma-Aldrich, St Louis, MO, USA), tissues samples (foregut, midgut, hindgut, heart, liver, spleen, kidney and muscle) were collected and immediately frozen in liquid nitrogen and stored at 80 C until further analysis. 2.2. RNA isolation and cDNA synthesis Total RNAs were isolated from the foregut, midgut, hindgut, heart, liver, spleen, kidney and muscle tissues by Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The integrity of the RNA was assayed by 1.2% agarose gel containing 1 pg/mL of ethidium bromide. The quantity of RNA was detected by spectrophotometer (BioPhotometer Eppendorf, Hamburg, Germany) based on A260/A280 ratio. Before the rst-strand cDNAs were synthesized, genomic DNA contamination was eliminated by incubation with DNase I for 60 min at 37 C (Fermentas, Vilnius, Lithuania). The rst strand cDNA synthesis was performed using 1 g total RNA and AMV reverse transcriptase (Fermentas) with the oligo (dT)1218 primer. 2.3. Cloning of the full length cDNA of PepT1 The PepT1 cDNA was cloned by PCR using degenerated primers designed based on conserved sequences of PepT1 in other teleosts (Table 1). Polymerase chain reaction (PCR) was performed as the following: for 30 cycles with 94 C for 30 s, 60 C for 30 s and 72 C for 90 s. The products were separated with 1% agarose gel. The DNA product was then cloned into pMD18-T (Takara, Japan) vector and sequenced. After obtaining the central core sequence of PepT1, the full length cDNA was isolated using the SMART RACE cDNA Amplication Kit (Clontech, Palo Alto, CA, USA). Specic nested PCR primers were designed based on the partial sequences (Table 1). For 3 RACE, two

Table 1 Primers used for PepT1 cloning and quantitative real-time PCR. Primer Degenerate PepT1+ Degenerate PepT1 GSP1+ GSP2 NGSP1+ NGSP2 PepT1 rt+ PepT1 rt -actin+ -actin Primer sequence 5-TCCATTAACGCTGGCAGT-3 5-GAGAAGTCSAGGCCGGT-3 5-TGCAGCTGGGCTTTGG-3 5-AACATTGGCAGAGGGATA-3 5-CACATGGCCTGGCAGAT-3 5-AAAGCCAGCGGGAAACAG-3 5-TGCTCTTGTTGTGTTCATCG-3 5-CTCTCTCTTGGGGTATTGCTT-3 5-GAACACTGTGCTGTCTGGAGGTA-3 5-CTTGGGTTGGTCGTTTGAATC-3 Usage CDS CDS 3 RACE 5 RACE 3 RACE 5 RACE Real-time Real-time Real-time Real-time

PCR PCR PCR PCR

amplications were performed under the same conditions: 94 C for 30 s, 62 C for 30 s and 72 C for 2 min with 30 cycles. For 5 RACE, the amplication conditions were: 94 C for 30 s, 60 C for 30 s and 72 C for 1 min with 30 cycles. 2.4. Quantitative real-time PCR The PepT1 mRNA levels were determined by quantitative real-time PCR in a Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The primers were designed using Primer Express 3.0 software (Table 1). -Actin was used as an endogenous control. Five microliters of rst-strand cDNAs (in a dilution of 1:20) as templates was added to 20 L PCR solution containing 20 nmol/L primers and 10 L SYBR Green PCR Master Mix (Applied Biosystems). For each sample, three repeats were performed with the following condition: 50 C 5 min and 95 C 10 min followed by 40 cycles at 95 C for 15 s and 60 C for 45 s. The corresponding real-time PCR efciency was calculated according to the equation: PCR efciency = (10 1/slope 1) 100%. The values detected from different amounts of RNA (5 times of series dilution) of the representative samples were parallel with the respective standard curve. Under these conditions, mean PCR efciencies were 101.2% and 98.8%, respectively, for the PepT1 and -actin. Following the amplication, the dissociation curve was done to verify if single product were generated. Negative RT control and negative NTC control were carried out to rule out DNA and/or dimer contamination. Relative mRNA expression was assessed using the 2 Ct method by SDS software v1.3.1. 2.5. PepT1 expression patterns in grass carp 2.5.1. Determination of PepT1 expression during early development Embryos and juvenile of grass carp were collected at different developmental stages (for each stage n = 5). Total RNAs were isolated with Trizol reagent and cDNAs were synthesized as described above. PepT1 mRNA expression levels were assayed by quantitative real-time PCR. 2.5.2. Determination of the tissue distribution of PepT1 transcripts RNAs were isolated from foregut, midgut, hindgut, heart, liver, spleen, kidney and muscle in adult grass carp. After synthesis of cDNAs, PepT1 expression in these tissues was assayed by quantitative real-time PCR. 2.5.3. Determination of the diurnal rhythm of PepT1 transcripts in grass carp intestine The intestine was divided into three sections: the foregut, midgut and hindgut. The RNAs were isolated from intestine tissues in grass carp (n = 5). PepT1 differential expressions of the tissues in grass carp were determined by quantitative real-time PCR.

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Z. Liu et al. / Comparative Biochemistry and Physiology, Part B 164 (2013) 194200 Table 3 Formulation of different dietary protein sources dietary. Group Wheat our Starch Soybean meal Fish meal Soy bean oil Fish oil Choline chloride Calcium hydrogen phosphate Chromium oxide Methyl cellulose Premix Nutrition indicators CP ME (kcal/kg) DE (kcal/kg) EE (%) CF (%) ASH (%) Fish meal 8.00 32.00 0.00 47.00 3.00 3.00 0.5 1.0 0.5 2.00 2.00 Soybean meal 8.00 12.00 68.00 0.00 2.00 3.00 0.5 1.0 0.5 2.00 2.00

2.6. Dietary protein levels and protein sources regulation on PepT1 To determine the effects of dietary protein levels on PepT1 expression, three isocaloric diets with different protein levels at 22, 32 and 42% crude protein (CP) were formulated (Table 2). Juvenile grass carp (0.5 years old) were kept in three berglass tanks (1.2 m H 0.8 m D) (n= 30 sh in each tank). Before starting the experiment, the sh were adapted to the experimental diets for 1 week. Fish were then fed with the three different diets with 22, 32 and 42% CP levels for 1 month, respectively. On day 7, day 14, day 21 and day 28, ve individuals were sacriced and the foreguts were collected. The levels of PepT1 mRNA expression were determined by real-time PCR in foregut. The effects of dietary protein sources on the grass carp PepT1 expression were analyzed by real-time RT-PCR. Two sh diets with the same amount of protein were formulated using sh meal or soybean meal (Table 3). The sh (0.5 years old) were kept in two berglass tanks (1.2 m H 0.8 m D) (n = 30 sh/tank) and adapted to the experiment diets in the experimental pond for 1 week before starting the experiment. These two groups of sh were fed with sh meal or soybean meal diets for 1 month, respectively. Every seven days, ve individuals were sacriced and the foreguts were collected. The transcripts of PepT1 mRNAs were assayed by real-time PCR. All diets were prepared by thoroughly mixing dry ingredients with oil and then adding water until a stiff dough resulted. The dough was then passed though a meat-mincer equipped with a 2 mm die, and the resulting strands were dried using an electrical fan at 28 C. After drying, the material was broken up into regular pieces sieved to a convenient pellet size and stored at 20 C. 2.7. The effect of sodium butyrate on PepT1 gene expression 2.7.1. In vivo study The sh were randomly divided into two groups: control and sodium butyrate group (n=30). The formula of control group contained 32% CP (Table 2) while the sodium butyrate group was added 0.1% sodium butyrate in the formula. The feeding conditions were the same as described above. Seven individuals were collected once a week. The total RNA of foregut was isolated and the PepT1 expression levels were assayed by real-time PCR. 2.7.2. In vitro study Primary cell culture was performed with grass carp intestinal cells to examine the effect of sodium butyrate on PepT1 expression in vitro. Three grass carp (2-years-old) were killed and the foreguts were separated by scissors. After washing three times with PBS, the organs

30.24 3211.00 3421.70 10.85 0.57 9.52

30.28 3102.60 3483.00 6.41 3.73 4.27

were incubated with 0.05% (w/v) collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min and followed by washing with PBS three times. Cells were cultured in 24-well culture plate with 1 mL DMEM containing 10% fetal bovine serum (Gibco BRL, MD, USA), at a density of 800 mg/each well. Cells were incubated in the cell culture incubator at 24 C with 5% CO2 for 2 days before sodium butyrate treatment. To examine the response to sodium butyrate in vitro, four different sodium butyrate concentrations (0 mM, 3 mM, 6 mM and 9 mM) were tested. Three replicates were done for each concentration. After a 12 h incubation, cells were harvested forPepT1 mRNA transcript analysis by RT-PCR. 2.8. Statistical analysis Data were analyzed by one-way analysis of variance. The normality and homogeneity of variance were tested by the Levene's test (Wei et al., 2012). If data did not have homogenous variance, they were log-transformed to meet the necessary assumptions of analysis of variance. Signicant differences among treatment means were determined by the Tukey's multiple range test. All statistical analyses were performed by SPSS 13.0 software (Chicago, IL, USA). P values b 0.05 were conrmed as signicant. 3. Results

Table 2 Formulation of different dietary protein levels. CP% Wheat our Starch Soybean meal Fish meal Soya bean oil Fish oil Choline chloride Calcium hydrogen phosphate Chromium oxide Methyl cellulose Premix Nutrition indicators ME (kcal/kg) DE (kcal/kg) EE (%) CF (%) ASH (%) 22% 8.00 37.00 32.00 12.00 3.00 2.00 0.5 1.0 0.5 2.00 2.00 32% 8.00 20.00 32.00 28.00 3.00 3.00 0.5 1.0 0.5 2.00 2.00 42% 8.00 3.00 32.00 44.00 3.00 4.00 0.5 1.0 0.5 2.00 2.00

3.1. Isolation and sequence analysis of PepT1cDNA from grass carp PepT1 cDNAs were isolated from a grass carp intestine cDNA library. The full length cDNA was 2762 bp (GenBank accession no. JN088166), with a 2142 bp open reading frame encoding a peptide of 714 amino acids. The 5 untranslated region (UTR) was 141 bp, while the 3 UTR was 479 bp long and contained a polyA tail. From the deduced protein sequence, seven putative extracellular N-glycosylation sites (Asn123, Asn449, Asn502, and Asn517) and one putative intracellular cAMP/ cGMP dependent protein kinase phosphorylation sites (Thr368, Ser704, and Ser720) were identied. A phylogenetic tree was constructed with the Mega 4.0 software using the Neighbor-Joining method (Fig. 1). The clustering pattern provides evidence that grass carp PepT1 is grouped with high bootstrap support in the lineage of other teleosts. It shares high sequence homology with goldsh (Carassius auratus) and zebrash (D. rerio) PepT1b. The PepT1 from mammals are grouped into two other distinct lineages (Fig. 1).

3282.40 3568.50 6.98 1.95 4.47

3192.60 3486.90 9.53 2.08 7.67

3102.80 3405.30 12.09 2.21 10.87

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Fig. 1. Phylogenetic tree of the PepT1 gene. The accession numbers are: GenBank: Carassius carassius ADK71159, Rattus norvegicus NP_476462, Mus musculus NP_444309, Xenopus (Silurana) tropicalis XP_002935692, Bos taurus NP_001092848, Oryctolagus cuniculus NP_001075806, Canis lupus familiaris NP_001003036, Equus caballus XP_001493109, Macaca mulatta NP_001028071, Pongo abelii XP_002824429, Homo sapiens NP_005064, Danio rerio 1b NP_932330, Fundulus heteroclitus macrolepidotus 1a AEX92274, F. heteroclitus macrolepidotus 1b AEX92273; Ensembl: D. rerio 1a ENSDART00000090215, Salmo salar NP_001140154, Oryzias latipes 1b ENSORLT00000003655, O. latipes 1a ENSORLT00000022074, Takifugu rubripes 1b ENSTRUT00000018670, T. rubripes 1a ENSTRUT00000044692, Tetraodon nigroviridis 1b ENSTNIT00000004977, T. nigroviridis 1a ENSTNIP00000016989, Gasterosteus aculeatus 1b ENSGACT00000018320 and G. aculeatus 1a ENSGACT00000005667.

3.2. PepT1 gene expression during early development of grass carp larvae PepT1 expression was analyzed by real-time RT-PCR during embryogenesis. PepT1 expression reached a maximum in the gastrula stage and a minimum in the organ stage. From the organ stage to 2 days after hatching, PepT1 mRNA levels gradually increased and reached a maximum on day 1 after hatching, and then quickly declined on day 2 after hatching. PepT1 expression reached maximum in 7 days after birth, and then gene expression remained at a constant levels from 14 days to 34 days in the juvenile (Fig. 2).

3.3. The temporal and spatial PepT1 gene expressions in various tissues of grass carp The tissue specic pattern of PepT1expression was analyzed in grass carp by quantitative real-time PCR. PepT1 mRNAs were expressed in the foregut, midgut, hindgut, liver, spleen, heart and muscle tissues. Higher levels of PepT1 expression were found in the foregut, liver, muscle, kidney, heart and spleen. Among all tissues expressing PepT1, foregut showed the highest levels of expression (Fig. 3).

Fig. 2. The relative abundance of PepT1 mRNA in different developmental stages. Error bars indicate the standard deviation. Different letters indicate statistical difference (P b 0.05).

Fig. 3. The relative abundance of PepT1 mRNA in different tissues. Error bars indicate the mean SD. Different letters indicate statistical difference (P b 0.05).

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Fig. 4. The relative abundance of PepT1 mRNA in circadian rhythm. Error bars indicate the mean SD. Different letters indicate statistical difference (P b 0.05).

To determine whether PepT1 was expressed at the same or different levels in various region of the intestine, the foregut, midgut and hindgut were dissected and PepT1 expression was analyzed by quantitative real-time PCR. The result showed that PepT1 mRNA levels were high in the foregut, low in the hindgut, and intermediate in the midgut. In addition, we analyzed the effect of day and night on PepT1 expression. The results revealed that PepT1 mRNA levels showed a diurnal variation in all three bowel segments (P b 0.05, for most of time) (Fig. 4). Expression of PepT1 was low in the daytime and high at night. 3.4. Effects of developmental age and dietary protein contents on PepT1 mRNA expression in the grass carp intestine The effect of sh diet on PepT1 expression was analyzed. After feeding with diets of different protein levels, PepT1 mRNAs expression was assayed by real-time PCR at 7 d, 14 d, 21 d and 28 d. At day 7, the PepT1 mRNA levels were the lowest (P b 0.05) in grass carp fed with the low-protein diet (22%), highest in grass carps fed with the 42% CP diet, and intermediate for those fed with the 32% CP diet (P b 0.05) (Fig. 5). However, grass carp fed with the 22% CP diet showed a signicant linear increase in PepT1 mRNA expression between day 7 to Day 28 (P b 0.05) (Fig. 5). In contrast, there were no signicant changes in the PepT1 expression prole in the group fed with the 32% CP diet (P > 0.05) (Fig. 5). Interestingly, sh fed

Fig. 6. Effects of developmental age and dietary protein source on PepT1 mRNA abundance in the grass carp intestine. Error bars indicate the mean SD. Different letters indicate statistical difference (P b 0.05).

with the 42% CP diet initially showed a decreased PepT1 mRNA abundance. However, PepT1 expression gradually increased with time from day 7 to day 28 (Fig. 5). Our data also indicate that protein

Fig. 5. Effects of developmental age and dietary protein level on PepT1 mRNA abundance in the grass carp intestine. Error bars indicate the mean SD. Different letters indicate statistical difference (P b 0.05).

Fig. 7. Butyrate increases PepT1 transcripts in vivo and in vitro. A. Dietary sodium butyrate promoted PepT1 expression in foregut of grass carp. B. Dietary sodium butyrate treatment increased PepT1 expression in foregut cells in primary culture. Error bars indicate the mean SD. Different letters indicate statistical difference (P b 0.05).

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source may have an effect on PepT1 gene expression. During the course of the four-week study, PepT1 mRNA abundance was higher in grass carp fed with sh meal than in carp fed with soybean meal (Fig. 6). 3.5. The sodium butyrate effects on PepT1 transcripts in the grass carp intestine The effect of sodium butyrate on PepT1 expression was analyzed in cultured intestinal cells from the grass carp. In vivo studies showed that the PepT1 mRNA levels were not signicantly altered initially by the sodium butyrate treatment (P > 0.05). However, long-term treatment with sodium butyrate could alter PepT1 transcripts levels with PepT1 gene expression enhanced over time in the treated group (P b 0.05) (Fig. 7a). Sodium butyrate increased PepT1 mRNA transcripts in a dose dependent manner (P b 0.05) (Fig. 7b). 4. Discussion In teleosts, PepT1 genes have been identied in several species, including zebrash (Verri et al., 2003), Atlantic cod (Rnnestad et al., 2007) and common carp (Ostaszewska et al., 2009). In the present study, we cloned a full-length cDNA from grass carp that encodes a PepT1-type transporter. Based on the deduced protein sequence, seven putative extracellular N-glycosylation sites and putative intracellular cAMP/cGMP dependent protein kinase phosphorylation sites were identied. It has been reported that protein kinase C could regulate the gene expression of PepT1 in Caco-2 cells (Ashida et al., 2002). Based on the conserved sites and domains, it is highly likely that the cloned cDNA indeed encodes PepT1 of grass carp. We also noticed that two PepT1 isoforms were found in several teleosts, including PepT1a and PepT1b (Bucking and Schulte, 2012). In present study, the phylogenetic tree shows that the cloned PepT1 of grass carp shared a high sequence identity with the PepT1b gene. Further studies are required to determine whether two isoforms of PepT1 are present in grass carp and goldsh. The PepT1 mRNA is strongly expressed in the small intestine and epithelium of kidney. Expression levels are especially high in the foregut. In contrast, mRNA expression is lower in colon, epithelium of bile duct, brain cells, liver and central kidney (Dring et al., 1998). It has been shown that in the vertical section of the intestine PepT1 expression is decreased from top of chorionic villus to the crypt site at the base (Takano et al., 2006), while the expression of PepT1decreases along the longitudinal axis from duodenum to ileum (Shen et al., 2001). This pattern of expression likely represents the regional difference of protein absorption. We demonstrated in grass carp that PepT1 was expressed in many tissues, including the foregut, midgut, hindgut, liver, kidney, muscle and heart with the highest levels of expression in the foregut, lower levels of expression in the mid-gut and the lowest expression in hind-gut. These results showed that the expression of PepT1 was decreased along the longitudinal axis of the intestine, consistent with previous ndings in chicken (Frazier et al., 2008), rainbow trout (Ostaszewska et al., 2010), loach (Gonalves et al., 2007) and European sea bass (Terova et al., 2009). During early sh embryonic development, nutrition is provided endogenously with the main protein source of sh fry derived from the yolk sac. After hatching, the fry rely on nutrients from exogenous food. Previous studies have shown that dietary proteins are nally digested as small peptides and the peptides are transferred from the extracellular compartment into the intracellular compartment by PepT1 (Daniel, 2004). Our results showed that during embryogenesis, PepT1 mRNA was expressed throughout all the periods with the highest levels of expression in the gastrula period. After hatching, the fry showed a stable pattern of expression from days 14 to 34. It is not clear whether the difference between endogenous and exogenous protein origins correlates with the different levels of PepT1

expression. Three days after hatching, the fry had a well developed digestive tract. This is followed by feeding ability developed between days 7 and 9. Thus, PepT1 expression increases with the development process in grass carp. This is consistent with previous data from Atlantic cod (Amberg et al., 2008) and chicken (Chen et al., 2005). Several studies reported a circadian rhythm of digestive enzymes and nutrient transporters in small intestine of mammals and fouls, which showed a signicant correlation with intestinal function. Data from these studies provide useful information for better diet composition and feeding management. In this study, we found the circadian rhythm of PepT1 in grass carp intestine with higher levels of expression during daytime than nighttime in all the studied tissues. Pan et al. (2004) reported that a diurnal rhythm of PepT1 expression was found in rats. In present study, we also showed a different level of PepT1 expression in grass carp between day and night which showed similarity with PepT1 expression of mammals. The mechanism for the circadian rhythm of PepT1 expression is not clear. It has been suggested by Pan et al. (2004), that the difference is caused by the time of food intake, rather than the light cycle. In this research, we showed that PepT1 was expressed at a higher level at night. The higher expression at night showed that the action of small peptide transfer was delayed after food intake which may due to the time elapse of protein digestion. Subsequently, di- and tri-peptides were transferred by PepT1 and the expression levels were increased in this process similar to the diurnal rhythm result in rats (Pan et al., 2002). We also analyzed PepT1 mRNA expression levels in sh of different ages fed with diets of different protein levels. Intriguingly, the dietary protein levels had different effect on PepT1 expression. The 22% CP diets had a positive effect on nPepT1 expression, while no signicant changes were found in sh fed with the 32% CP diets. However, in 42% CP diets group, a negative correlation was found between days 14 and 21 of feeding, suggesting that diets with different protein levels could affect the expression of PepT1 mRNA in carp. Moreover, high level of protein diets may not the most suitable for sh aquaculture. In recent studies, a signicant correlation has been demonstrated between PepT1 expression and animal growth rate (Matsumura et al., 2005; Barrenetxe et al., 2006; Ostaszewska et al., 2010). The knowledge of nutrient transport has been regarded as important information for development of new formula of commercial diet to enhance sh growth rate. As we found, the 22% CP diet showed a strong positive correlation with PepT1 expression. Strikingly, the 22% CP group had the highest levels of PepT1 expression when analyzed at 28 day compared with two other diets with higher protein levels. These data indicate that lower protein diets may have a better stimulation on PepT1 expression and transfer of small peptides. As a novel feed additive that can be used for animals, sodium butyrate could regulate electrolyte balance and gastrointestinal tract micro-ecology balance, enhance the function of digestion and absorption of small intestine (Salminen et al., 1998). Hence it can improve the health and growth performance of animals and increase commercial benets for farmers. Though grass carp is a representative and native Chinese freshwater sh, feed additive sodium butyrate has not been used in feed formulation. We demonstrated in this study that sodium butyrate could increase PepT1 expression in the grass cap intestine in vivo and in vitro, consistent with previous studies in Caco-BBE cell (Yamashita et al., 2002). We inferred that feed additive sodium butyrate can regulate the transport of protein digestion products (di- and tri-peptides), and may have useful applications in stimulating protein absorption for grass carp in aquaculture. In summary, PepT1 of grass carp has been cloned and sequenced. The cDNA and deduced protein share a high sequence similarity with PepT1 of other teleosts. The expression pattern of PepT1 has been determined in grass carp during early development and in various adult tissues. Moreover, we found that the dietary protein levels had a signicant effect on PepT1 expression. Furthermore, we found that sodium butyrate stimulated the expression of PepT1 which

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could induce faster growth rate in grass carp. The regulation of PepT1 expression by protein levels, sources and sodium butyrate could provide new clues to help us design more suitable formula of sh diets for grass carp in aquaculture. Acknowledgments This research was supported by the National Natural Science Foundation of China for Young Scholars (Grant No. 31001114) and Hunan Provincial Natural Science Foundation of China (Grant No. 12JJ6018). References
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