http:// www.jstage.jst.go.jp / browse / jpsa doi:10.2141/ jpsa.0110125 Copyright Ⓒ 2013, Japan Poultry Science Association. Molecular Cloning, Characterization, and mRNA Expression of Intestinal Fatty Acid Binding Protein (I-FABP) in Columba Livia Peng Xie, Lian-Long Liu, Chao Wang and Xiao-Ting Zou Key Laboratory for Molecular Animal Nutrition of Ministry of Education, Feed Science Institute, Zhejiang University, Hangzhou 310058, China Intestinal fatty acid binding protein (I-FABP) is involved in fatty acid transportation in mammals. To verify its role in fatty acid metabolism in birds, the model of pigeon intestinal organ culture was established, and a full-length cDNA of I-FABP was cloned from the intestine of Columba livia by rapid amplification of cDNA ends (RACE) method for the first time. The full length cDNA of C. livia I-FABP was 855 bp, including a 5′ untranslated region (UTR) of 34 bp, a 3′ UTR of 422 bp and an open reading frame (ORF) of 399 bp encoding a protein of 132 amino acids with the predicted molecular weight of 15.13 kDa. Sequence comparison indicated that I-FABP of C. livia had high identity with other avian I-FABP and belonged to the first subfamily of FABPs. Using quantitative real-time PCR, pigeon I-FABP mRNA in duodenum and jejunum increased progressively with development stage. I-FABP mRNA was expressed at the highest level at 28 day post hatch in the duodenum, while in ileum, it reached the maximum at the day of hatch, and decreased subsequently. The effects of fatty acids on pigeon I-FABP expression were also investigated in vitro. The results showed that significant increases in the pigeon I-FABP mRNA level were induced by linoleic acid and arachidonic acid, whereas I-FABP gene expression appeared to be unaffected by oleic acid and αlinolenic acid. The results indicate that I-FABP may be indicative of intestine development in pigeon, and it could be regulated by n-6 polyunsaturated fatty acids. Key words: cDNA cloning, Columba livia, intestinal fatty acid binding protein, long-chain fatty acids, mRNA expression J. Poult. Sci., 50: 9-19, 2013 Introduction Cytosolic fatty acid binding proteins (FBAPs) are low molecular weight (14-15 kDa) proteins with high affinities for hydrophobic ligands, such as long-chain fatty acids (LCFAs) (Veerkamp and Maatman, 1995). To date, at least 10 FABP isoforms have been identified in vertebrates (Bernlohr et al., 1997). According to the initial tissue from which they are first isolated, members of FABPs are named as followed: Adipocyte-type FABP (A-FABP), Myelin P2 protein (MP2), Testis-type FABP (T-FABP), Epidermaltype (E-FABP), Brain-type (B-FABP), Heart-type (HFABP), Intestinal-type (I-FABP), Liver basic-type (LBFABP), Ileal-type (IL-FABP) and Liver-type (L-FABP). Similar with other FABP isoforms, tertiary structure of IFABP consists of two short α-helices and 10 antiparallel βReceived: October 18, 2011, Accepted: June 10, 2012 Released Online Advance Publication: July 25, 2012 Correspondence: Prof. XT Zou, Key Laboratory for Molecular Animal Nutrition of Ministry of Education, Feed Science Institute of Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China. (E-mail: [email protected]) strands, which organize into a helical cap and two orthogonal β-sheets (Bernlohr et al., 1997; Marcelino et al., 2006). LCFAs can be bound within the barrel in the central of internal water filled cavity. Due to this protein specially found in mature enterocytes, I-FABP was thought to be crucial in fatty acids trafficking, and targeting ligands to specific organelle for metabolic process. Even so, the specific function of I-FABP in animal intestine remains elusive. LCFAs play central roles in human and animal growth and development. Not only do they provide sources of metabolic energy, components of membranes, or precursors for eicosanoids and docosahexenoic acid, but also serve as ligands or transcription factors in cell signal transduction and gene expression regulation (Innis, 2007; Woods and Fearon, 2009). Studies focused on mammals have shown that dietary regulation can affect I-FABP expression. Rats fed with highfat diets containing 20-38% vegetable oil showed a 40-50% increase in cytosolic content of I-FABP (Ockner and Manning, 1974). Conversely, reports also show that I-FABP expression increases by 100% after 3 days of feed deprivation but remains unaltered by a 7-day fat feeding in the mouse (Besnard et al., 1991; Poirier et al., 1997). However, in- Journal of Poultry Science, 50 (1) 10 formation about avian I-FABP is very scarce. Only three avian I-FABP sequences have been obtained, including chick (Gallus gallus), duck (Anas platyrhynchos) and zebra finches (Taeniopygia guttata). Knowledge on the developmental pattern of I-FABP expression and its regulation mechanism in birds is rarely reported. The pigeon (Columba livia), a typical representative of altrices, is widely bred for its meat, use in sport, and as an experimental animal in behavioural or viral experiments (Sales and Janssens, 2002). However, very little research about the development mechanism and nutrient metabolism has been studied in this species. Therefore, in the present study, we first cloned the I-FABP gene in pigeons, and characterized the gene expression from the embryonic period to 28-day squab. Furthermore, we evaluated different LCFAs on the expression profile of I-FABP in an organ culture system. The identification and characterization of I-FABP may contribute to a better understanding of fatty acid metabolism in avian species and develop useful strategies for nutrient requirements in the pigeon industry. Materials and Methods The experiment was conducted in accordance with Chinese guidelines for animal welfare and was approved by the animal welfare committee of the Animal Science College, Zhejiang University. Animals and Embryos Twenty-four healthy young pigeons (C. livia), were obtained from Xingliang commercial pigeon farm (Wenzhou, China). To study the embryogenetic expression profiles, fertile eggs were also obtained from the same farm. They were weighed and 24 of them were divided into four groups with equal weight frequency distribution, average egg weight was 18.4×1.2 g. The remaining eggs were used for intestine organ culture. The eggs were incubated under optimal condition (38.1℃, 55% relative humidity) in the incubator. Six young pigeons at post-hatching day 3 (3d), 8 (8d), 14 (14d) and 28 (28d) were killed. The eggs were opened at the blunt end using surgical scissors, and the embryos from each group were sacrificed to be sampled on the following days: embryonic day 11 (11e), embryonic day 13(13e), embryonic day 15 (15e) and hatching day (0d). For each pigeon and embryo, the small intestine was dissected and divided into three parts, duodenum, jejunum and ileum. They were quickly frozen in liquid nitrogen and then stored at −80℃. Organ Culture Tissue culture of pigeon embryonic intestine was conducted by the methods described previously with some modifications (Shimizu et al., 1991; Chen et al., 1994). Briefly, the jejunum was excised from the 14-day pigeon embryo and the mesentery was removed. The embryonic jejunum was cut into 2 mm long segments, and immediately washed twice with calcium- and magnesium-free Hanks balanced salt solution (HBSS, M&C Gene, China). The fragments were placed on a rectangular grid of 60-mesh stainless steel in the 1.5 cm-radius central well of a plastic culture dish (BD Bioscience, USA). Then 1 ml culture me- dium was added into the well. The serum-free culture medium was composed of a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM; Invitrogen, USA) and Roswell Memorial Institute 1640 medium (RPMI 1640; Genom, China), supplemented with 10 mM HEPES, 2 mM glutamine, and 0.2% fatty acid-free bovine serum albumin (BSA; Wako, Japan). In order to maintain a humid environment, a ring of sterile filter paper saturated with 150 mmol/l NaCl solution was placed surrounding the central well. The cultures were incubated at 37℃ in a 95% O2 and 5% CO2 atmosphere. Enzyme Activities To testify the availability of intestinal organ culture system, jejunum samples collected from 0 to 7d during the incubation were homogenized in lysis buffer (KeyGEN, China) with an Ultra-Turrax (T8, IKA-Labortechnik, Staufen, Germany). This process was conducted on ice. The homogenate was centrifuged at 10,000 rpm for 1 min at 4℃, and the supernatant was used for measuring the activities of caspase3 enzyme, alkaline phosphatase (AKP) and succinate dehydrogenase (SDH). The caspase-3 enzyme was examined by assay kit purchased from KeyGEN Company, and AKP, SDH and protein content in samples were determined by assay kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, Jiangsu, China). Preparation of Fatty Acid Sodium Salt and Fatty Acid-BSA Complex Preparation of fatty acid sodium salt and fatty acid-BSA complex was made according to the method of Alvaro et al. (2010). Briefly, 10 mg of fatty acids (oleic acid, linoleic acid, α-linolenic acid, palmitic acid and arachidonic acid) (Sigma-Aldrich, St. Louis, USA) were mixed with 0.5 ml EtOH and 5 M NaOH was used to adjust the molar ratio of fatty acids to NaOH 1:1. The mixture was dried under nitrogen gas and then dissolved in 2 ml of sterile water to make the stock solutions of fatty acids. 1 μM butylated hydroxytoluene was added to the stock solutions in order to prevent oxidation. 5 mM fatty acid free BSA was used to combine with the fatty acids, the molar ratio of fatty acid to BSA was 3:1, the fatty acid-BSA solutions were sterilefiltered and used fresh. RNA Isolation and Reverse Transcription Total RNA was extracted from the intestines using Trizol reagent (Invitrogen) and genomic DNA was eliminated using RNase-free DNase (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The quality of total RNA was checked by both native RNA electrophoresis on 1.0% agarose gel and the UV absorbance ratio at 260 nm and 280 nm. cDNA was synthesized by M-MLV reverse transcriptase (TaKaRa) at 42℃ for 60 min with oligo dT-Adaptor primer (Table 1) following the protocol of the manufacturer. Cloning the Full-length cDNA of C. livia I-FABP To identify I-FABP partial coding sequences, degenerated primers IF-F, IF-R (Table 1) were designed based on the highly conserved sequences of I-FABP from Gallus gallus (NP_001007924), Taeniopygia guttata (XP_002187542), Anas platyrhynchos (JN202313), Xenopus laevis (AAI 70117), Danio rerio (NP_571506), Sus scrofa (AAX62516), Xie et al.: I-FABP in Pigeon Table 1. Primer name IF-F IF-R 5′CDS-primer 3′CDS-primer SMARTer II A Oligonucleotide UPM 11 Primer used in the present study Primer sequence (5′ -3′ ) AGRAARYTDGSAGCYCAC TCHGTYCCRTCWGCBAGA (T)25VN AAGCAGTGGTATCAACGCAGAGTAC(T)30VN AAGCAGTGGTATCAACGCAGAGTACXXXXX GSP1 GSP2 IF-QF IF-QR 18S-QF 18S-QR Oligo dT Long: CTAATACGACTCACTATAGGGCAA GCAGTGGTATCAACGCAGAGT Short: CTAATACGACTCACTATAGGGC GTGATCTTCAGATTATCGTGGGCTCC GGAGCCCACGATAATCTGAAGATCAC AGAGAAAGTTAGGAGCCCACGA ACCAGTAAGTTCAGTCCCATCAG AGCTCTTTCTCGATTCCGTG GGGTAGGCACAAGCTGAGCC TTTTTTTTTTTTTTTTTT M13-47 RV-M CGCCAGGGTTTTCCCAGTCACGAC GAGCGGATAACAATTTCACACAGG Application Degenerate primersa For RACE-PCRb For quantitative real-time PCR For reverse transcription Vector primer RACE: rapid amplification of cDNA ends a R, A/G; Y, C/T; D, A/T/G; S, C/G; H, A/T/C; W, A/T; B, T/G/C b N, A/T/G/C; V, A/G/C; X: undisclosed base in the proprietary SMARTer oligo sequence Rattus norvegicus (EDL82110). The PCR was performed in a total volume of 25 μL on Thermal Cycler (Longene, China), PCR mixture contained 2 μL of 2.5 mmol/L dNTP mix, 2.5 μL 10×reaction buffer, 2 μL of 25 mM MgCl2, 2 μL of each primer IF-F and IF-R, 2 μL template cDNA, 0.25 μL of ExTaq DNA polymerase (5 U/μL) (TaKaRa), and 12.25 μL of MilliQ water. RT-PCR cycles were conducted at 95℃ for 3 min followed by 35 cycles of 94℃ for 30 s, 55℃ for 1 min, 72℃ for 1 min, and a final cycle of 72℃ for 10 min. The PCR products were analyzed by electrophoresis on 1.2% agarose gel, and then purified by DNA purification kit (TaKaRa). The products were integrated into pMD 18-T vector (TaKaRa). Vectors containing cloned inserts were transformed into E.coli DH5α and incubated for 12 hours at 16℃. Positive clones were identified by blue/white screening and PCR screening with M13-47 and RV-M primers (Table 1), and then sequenced. Full length of pigeon I-FABP cDNA was obtained by the method of rapid amplification of cDNA ends (RACE) technology. The SMARTTM RACE cDNA Amplification Kit (Clontech, USA) was used to perform RT and RACE PCR. The synthesis of the first strand cDNA was conducted using reverse transcriptase with SMARTer II A Oligonucleotide and 5′ -CDS primer A for 5′ RACE and 3′ -CDS primer A for 3′ -RACE. Reaction conditions were followed by the manufacturer’s instruction. For 5′ -RACE, the primer GSP1 and the universal primer A mix (UPM) were used. For 3′ -RACE, the primer GSP2 and UPM were used. All these primers are listed in Table 1. The PCR reaction conditions were conducted by the manufacturer’s protocol in the AdvantageTM 2 PCR kit (Clontech, USA). Sequence Analysis The open reading frame (ORF) of I-FABP gene was obtained by ORF Finder (http: //www. ncbi. nlm. nih. gov/gorf/ gorf.html). The protein molecular mass and isoelectric point (pI) of pigeon I-FABP were predicated by ExPASy Compute pI/Mw tool (http: //web. expasy. org/compute_pi/). The homology search of nucleotide and protein sequence of pigeon I-FABP was using BLAST program (http://www. ncbi.nlm. gov/blast). PROSITE program was performed to analyze the functional sites of deduced amino acid sequence (http:// www.expasy.org/prosite). The domain analysis was using SMART tool (http://smart.embl-heidelberg.de/smart/set_ mode.cgi?NORMAL=1). Multiple Sequences Alignment and Phylogenetic Analysis Multiple sequence alignment of the I-FABP was performed with software ClustalX 2.0. The result was viewed on BoxShade Server. A neighbor-joining phylogenetic tree was constructed using MEGA software version 4.0. The reliability of branching was tested by bootstrap analysis which performed for values representing 1000 replicates. I-FABP mRNA Expression in Embryonic and Post-hatching Pigeon Small Intestine Small intestines of fertilized embryos and squabs from different development stages (including 11e, 13e, 15e, 0d, 3d, 8d, 14d and 28d) were sampled to dissect into three parts, duodenum, jejunum and ileum, and I-FABP mRNA expressions were separately detected by SYBR Green quantitative real-time PCR (qRT-PCR). The method for total RNA isolation and reverse transcription was the same as described in section 2.4. The qRT-PCR was performed on an ABI StepOne Plus Real-Time PCR system (Applied Biosystems, USA). Two Journal of Poultry Science, 50 (1) 12 gene specific primers IF QF and IF QR (Table 1) were used to amplify a 161 bp I-FABP gene fragment and two 18S primers 18S QF and 18S QR (Table 1) were used to amplify a 256 bp 18S gene fragment as the internal control. PCR reaction used SYBR Premix PCR kit (TaKaRa). The PCR program was 95℃ for 30 s, followed by 42 cycles of 95℃ for 3 s, 60℃ for 10 s and 72℃ for 30 s. The standard curve was determined using pooled samples. Each sample was performed in duplicate and negative controls without cDNA template were included in this experiment. Specificity of the amplification was verified at the end of PCR run by melting curve analysis. The relative expression quantity was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001). I-FABP mRNA Expression in Pigeon Jejunum After Fatty Acids Incubation In vitro To test if fatty acids may affect the expression of I-FABP, pigeon embryonic jejunums were treated by different concentrations (5, 50 and 250 μM) of fatty acids in organ culture system for 24 h. The tissues were then collected to analyze the I-FABP mRNA expression. The methods of total RNA extraction and qRT-PCR were the same as those described above. The primers of I-FABP and 18S for were also the same. Each treatment had three replicates. Statistical Analysis All data were presented as means±S. E. of the relative mRNA expression. The data were statistically analyzed by one-way ANOVA by SPSS 17.0 (SPSS Inc., Chicago, IL), and the level of statistically significant difference was set at P<0.05. Results Changes of Enzyme Activities in Pigeon Jejunum During the Period of Organ Culture To investigate the availability of intestinal organ culture system, enzyme activities of caspase-3, AKP and SDH were determined from 0 to 7 d during the culture. As shown in Fig. 1, caspase-3 activity was increased significantly at 1 d (P<0.05), but it decreased subsequently and remained the same level between days 2 and 7 except that at 6 d. No differences were observed in AKP activity between days 0 and 2, but it increased significantly during the culture period at day 3-7 (P<0.05). SDH activity between days 2 and 6 was significantly higher than that at 0 and 1 d (P<0.05). Cloning and Identification of Pigeon I-FABP cDNA The full-length cDNA of pigeon I-FABP was obtained by the RACE method. The complete sequence of I-FABP cDNA was 855 bp, containing a 5′ untranslated region (UTR) of 34 bp, a 3′ UTR of 422 bp and an ORF of 399 bp (Fig. 2). The ORF encoded 132 amino acid residues including an 18 amino acids cytosolic fatty-acid binding proteins signature. The calculated molecular mass of the protein was 15.13 kDa with theoretical pI of 6.75. The sequence of I-FABP was submitted to NCBI under GenBank accession number JF835078. Multiple Sequences Alignment and Phylogenetic Analysis As shown in Fig. 3, the deduced amino acids of pigeon IFABP shared high identity with reported I-FABP in other Activity of caspase-3 enzyme, alkaline phosphatase and succinate dehydrogenase in the pigeon embryonic jejunum at 0, 1, 2, 3, 4, 5, 6, 7 d of culture in vitro. Data are means±S.E. (n=3). Fig. 1. species. Blast search indicated that pigeon I-FABP presented 91% identity with A. platyrhynchos, 87% with G. gallus, 80% with T. guttata, 79% with Mus musculus, 76% with Homo sapiens. 18 amino acids, from residue 5 to 22, were identified as a cytosolic fatty-acid binding proteins signature in most species shown in Fig. 3 except Equus caballus. A lipocalin domain was found in pigeon I-FABP within the amino acids sequence started at position 4 and ended at position 131, which was the same as it in A. platyrhynchos, while those of other species all covered from position 4 to position 132. To reveal the molecular phylogenetic position of pigeon IFABP, a phylogenetic tree of reported FABPs’ amino acid sequences was constructed by neighbor-joining method from Xie et al.: I-FABP in Pigeon 13 Nucleotide and deduced amino acid sequence of intestinal fatty acid binding protein of pigeon C. livia (Genbank accession number JF835078). The cytosolic fatty-acid binding proteins signature is underlined letters. Polyadenlation signal is double-underlined. The asterisk indicates the stop codon. Fig. 2. a distance matrix, performed with the MEGA 4.0 software (Fig. 4). Two subfamilies of FABPs were found. The adipocyte, testis, epidermal, brain, heart and intestinal FABP and myelin P2 protein were confined to the first group. The second group contained liver basic, ileal and liver FABP. Pigeon I-FABP belonged to first subfamily. Embryogenetic and Post-hatching I-FABP Expression in Pigeon The development patterns of I-FABP gene expression in duodenum, jejunum, and ileum were analyzed by quantitative real-time PCR. As shown in Fig. 5, a similar expression trend was found in duodenum and jejunum, I-FABP mRNA level increased with progressing of development stage, and both of them reached a peak value at post-hatching 28 day (28d) (P<0.05). However, an inverse expression trend was found in the ileum, I-FABP gene expression reached a maximum at the hatching day (0d) (P<0.05), but then gradually decreased to embryonic level. Effects of Fatty Acids on I-FABP mRNA Expression To investigate whether I-FABP expression was regulated by LCFAs, jejunum fragments in an organ culture system were treated with oleic acid, linoleic acid, α-linolenic acid, palmitic acid and arachidonic acid at the concentrations of 5, 50 and 250 μM respectively. The organs were incubated with those LCFAs for 24 hours and I-FABP mRNA expression was determined. As shown in Fig. 6, oleic acid and αlinolenic acid had no effect on I-FABP mRNA expression (P >0.05) compared with the control group, whereas the addition of 5 and 50 μM linoleic acid significantly increased I- FABP mRNA level by 146% and 95% (P<0.05) respectively. However, when 250 μM linoleic acid and palmitic acid was used, I-FABP mRNA level was decreased by 52% and 59% (P<0.05) respectively. In contrast, high concentrations of arachidonic acid appeared to have greater inducement since 250 μM of which can significantly increase the I-FABP mRNA level by 150% (P<0.05). Discussion Organ culture of intact small intestine had been proved to be a useful technique for the study of virus pathogenicity, drug and nutrient metabolism or cell differentiation (Corradino, 1974; Batt et al., 1995; Tou et al., 2004). Kedinger et al. (1974) reported that organ culture of adult guinea-pig intestine can just be available for 24h on the condition that the villi remained undamaged. However, the gut segments from mouse embryo were grown in catenary culture for up to 10d (Hearn et al., 1999). So, it is suggested that organ culture of animal embryonic intestine seemed to have much stronger activity than that of adult animal. In the current study, we investigated the activity of caspase-3 which is a key enzyme in regulation of cell apoptosis, the results indicated that the intestine extracted from the body cultured in vitro may require an acclimation. ALP, expressed by the enterocytes on the top of the villi, is a marker of mucosal enterocyte maturation (Weiser, 1973; Traber et al., 1992). SDH is a membrane-bound dehydrogenase linked to the respiratory chain and a key member of the tricarboxylic acid (TCA) cycle. Continuous increase of these two enzymes Journal of Poultry Science, 50 (1) 14 Multiple alignments of pigeon C. livia I-FABP with other known I-FABP deduced amino acid sequence: Gallus gallus (NP_ 001007924), Anas platyrhynchos (JN202313), Taeniopygia guttata (XP_ 002187542), Xenopus laevis (AAI70117), Danio rerio (NP_571506), Salmo salar (ACI69121), Homo sapiens (AAI11792), Sus scrofa (AAX 62516), Equus caballus (AAT08114), Mus musculus (NP_032006), Rattus norvegicus (EDL82110). Cytosolic fatty-acid binding proteins signature is enclosed with brace above the alignment. Asterisk indicates amino acid identity. I-FABP=intestinal fatty acid binding protein. Fig. 3. indicated that the pigeon embryonic gut segments in vitro still maintained high rate of metabolism during the 7d culture. Therefore, the intestine organ culture system used in this study was available, and it was also suggested that 24 h should be pre-cultured before the experiment began. Intestinal fatty acid binding protein (I-FABP) is cytosolic protein synthesized in the polarized columnar epithelial cells (Scapin et al., 1991). It has been thought to play an important role in shuttling fatty acids, because of its internal site for non-covalent binding of long-chain fatty acids (Zimmerman and Veerkamp, 2002). In the present study, we used genespecific primers to clone and analyze the pigeon I-FABP gene sequence, the full length of pigeon I-FABP cDNA reported encoded a putative fatty acid binding protein of 131 amino acids, which was the same as that in mammals and other birds (Green et al., 1992; Wang et al., 2005; Jiang and Li, 2006). Cytosolic fatty-acid binding proteins signature from the N-terminal extremity and a lipocalin domain were also detected. On the basis of similarities of structure, function and sequence, this family with other two, the lipocalin and avidin/streptavidin families, form a larger group: the calycin structural superfamily (Flower, 1993; Flower et al., 1993). The clustalX alignment of pigeon I-FABP revealed high identity with avian I-FABP sequences (80%-90%), but Xie et al.: I-FABP in Pigeon 15 Fig. 4. Neighbor-joining phylogenetic tree of FABP amino acid sequences from selected species. The Genbank accession numbers for the sequences are as follows: A-FABP, Homo sapiens (NP_001433), Mus musculus (NP_077717), Gallus gallus (NP_989621), Anas platyrhynchos (AEH03009), Anser anser (AAl79836), Taeniopygia guttata (XP_002199746), Danio rerio (ABB96216), Salmo salar (ACN12235); B-FABP, Homo sapiens (NP_001437), Mus musculus (AAA81904), Gallus gallus (NP_ 990639), Taeniopygia guttata (XP_002188863), Danio rerio (NP_999972), Salmo salar (ACN12396); E-FABP, Homo sapiens (NP_001435), Mus musculus (NP_034764), Sus scrofa (ABD18458); H-FABP, Homo sapiens (NP_004093), Mus musculus (NP_ 034304), Gallus gallus (NP_001026060), Anas platyrhynchos (ABH10617), Danio rerio (NP_694493), Salmo salar (ACI69906); I-FABP, Homo sapiens (AAI11792), Mus musculus (NP_032006), Gallus gallus (NP_001007924), Anas platyrhynchos (JN202313), Taeniopygia guttata (XP_002187542), Danio rerio (NP_571506), Salmo salar (ACI69121); IL-FABP, Homo sapiens (EAW61565), Mus musculus (EDL33855), Danio rerio (AAH72553), Salmo salar (ACI68068); LB-FABP, Anas platyrhynchos (HM049632), Gallus gallus (AAK58094), Taeniopygia guttata (XM_002196411), Danio rerio (AAF67743), Rhamdia sapo (P80856), Homo sapiens (NP_001434), Mus musculus (AAH09812), Gallus gallus (AAK58095), Taeniopygia guttata (XP_002188068), Anas platyrhynchos (AEH03008), Danio rerio (NP_001038177), Salmo salar (NP_001134229); MP2, Homo sapiens (P02689), Mus musculus (P24526), Sus scrofa (P86412); T-FABP, Homo sapiens (NP_001073995), Mus musculus (AAH48437), Bos Taurus (NP_001179339). FABP=fatty acid binding protein. A-FABP=adipocyte-type fatty acid binding protein. MP2=myelin P2 protein. T-FABP=testis-type fatty acid binding protein. E-FABP=epidermal-type fatty acid binding protein. B-FABP=brain-type fatty acid binding protein. H-FABP=heart-type fatty acid binding protein. I-FABP= intestinal-type fatty acid binding protein. LB-FABP=liver basic-type fatty acid binding protein. IL-FABP=ileal-type fatty acid binding protein. L-FABP=liver-type fatty acid binding protein. 16 Journal of Poultry Science, 50 (1) Fig. 5. Gene expression of intestinal fatty acid binding protein in duodenum, jejunum, and ileum of C. livia during embryogenesis and post-hatching stage. Gene expression was analyzed by quantitative of real-time PCR. The development stage included embryonic day: 11e, 13e, 15e; hatching day (0d); post-hatching day: 3d, 8d, 14d and 28d. Vertical bars represent the means±S.E. (n=6 embryos or squabs per group) and bars with different letters (a, b and c) are significantly different (P<0.05). low identity (26%-31%) with other types of avian FABPs (data not shown). Phylogenetic analysis in this study suggested that two subfamilies of FABPs diverged from a common progenitor as previously reported (Jiang and Li, 2006; Gong et al., 2010), which was arose by gene duplications that occurred prior to the most recent common ancestor of mammals and bony fishes (Hughes and Piontkivska, 2011). Schaap et al. (2002) reported that I-FABP and other three FABPs (E-FABP, H-FABP and B-FABP) diverged from a common ancestral gene about 850 million years ago. Pigeon I-FABP belonged to the first subfamily of FABPs, and was more similar to that in A. platyrhynchos and G. gallus. Minimal mRNA expression of intestinal fatty acid binding protein was detected in pigeon embryonic intestine, while a sharp increase, 1115 fold, 512 fold and 229 fold in I-FABP expression can be found respectively in duodenum, jejunum, and ileum at the day of hatch. Similar results were also reported in chick and turkey, I-FABP-binding activity showed significant increases after hatch (Katongole and March, 1980; Ding and Lilburn, 2002). But in mouse, the level of IFABP mRNA rises rapidly on embryonic day 17 (Green et al., 1992). It is thought that the intestine in birds is still an immature organ at hatching and the expression of fatty acid binding protein could be representative of many physiological processes that must develop post hatch before digestive function becomes mature (Ding and Lilburn, 2002). In addition, the increase in I-FABP may be in concert with the uptake of dietary fatty acids. Unlike the chick and other poultry, newly hatched pigeon squabs are fed pigeon “milk”, called crop milk from parents for nearly 28d. It has been reported that crop milk contains high concentrations of fat and protein in the first five days after squabs hatching, and fat is the principle energy source in squabs diet, which in turns cause an 8-fold increase in body weight in the first week (Shetty and Hegde, 1991). So, it is suggested that the development rate of function of intestinal fatty acid transportation is very rapid. However, I-FABP expression in the ileum declined with the developmental progression, which was different from that in duodenum and jejunum. In mice, IFABP has a horizontal gradient (duodenal to colon) of expression, with the highest levels of expression in the jejunum and ileum (Veerkamp and Maatman, 1995). The reason for this difference may be partially attributed to the distinct intestinal structure of flying birds. In order to maintain the high level of metabolism and decrease the flying load, the digestion rate of pigeons is rapid, not only are the caeca degenerated but the colon and rectum are very short. It is likely that fatty acid is transported more efficiently in pigeon duodenum and jejunum. I-FABP expression in the ileum was higher in the early stage, which may indicate that ileum just plays a supplementary role in fatty acid transportation. Intestinal fatty acid binding protein has been thought to play an important role in intracellular transport of fatty acids. However, studies focused on the relationship between IFABP expression and dietary fat often get inconsistent results (Ockner and Manning, 1974; Poirier et al., 1997). In crop milk, oleic acid comprises nearly one half of the total fatty Xie et al.: I-FABP in Pigeon 17 Regulation of pigeon I-FABP mRNA level by fatty acids of various chain carbon length and saturation. Jejunum fragments were incubated for 24 h with increasing concentrations (5, 50 and 250 μM) of oleic acid, linoleic acid, α-linolenic acid, palmitic acid and arachidonic acid separately. I-FABP mRNA expression was determined by quantitative real-time PCR. Vertical bars represent the means±S.E. (n=3) and bars with different letters (a, b and c) are significantly different (P< 0.05). Fig. 6. acids, and palmitic acid is the commonest saturated acid in the triglycerides, which accords for approximately 16%, and then linoleic acid accords for 13%, α-linolenic acid and arachidonic acid were the least, no less than 1% (Desmeth, 1980). In spite of their high concentrations in crop milk, oleic acid and palmitic acid can not induce the increase of pigeon I-FABP gene expression in our study, which was different from the results of liver-FABP found in rat (Meunier-durmort et al., 1996). Overexpression of I-FABP gene also discloses a low rate of oleic acid incorporation but shows a significantly increase the level of mitochondrial fatty acid oxidation (Montoudis et al., 2006, 2008). This could be another proof that, like I-FABP in mammals (Vassileva et al., 2000), pigeon I-FABP may also function as a lipid-sensing component but is not essential for fat absorption. In addition, our study found that pigeon I-FABP mRNA level had close relationships with linoleic acid and arachidonic acid. Low concentration of linoleic acid and high concentration of Journal of Poultry Science, 50 (1) 18 arachidonic acid all could increase the expression of IFABP. So, pigeon I-FABP expression was not sensitive to those fatty acids accord for high concentrations in crop milk, and I-FABP expression may be different when animals were fed diets with different fatty acid composition. Peroxisome proliferator activated receptors (PPARs) play a key role in lipid metabolism (Issemann and Green, 1990; Palmer et al., 1998), which are found to be candidates for transcription of liver-FABP (Poirier et al., 2001). Gene structure analysis also show that I-FABP in mammals contain putative PPAR binding element (DR-1) at upstream region (Sweetser et al., 1987; Green et al., 1992), which suggests that I-FABP expression may be regulated by the factors which can interact with PPARs. Meanwhile, the relative potency for PPAR activation has been compared among the fatty acids as followed: linoleic acid > arachidonic acid > oleic acid, and saturated fatty acids showed the lowest affinity with the receptor (Jump and Clark, 1999). Therefore, it could explain our founding that I-FABP expression in pigeon was prone to be affected by linoleic acid at lower concentration compared with arachidonic acid, and oleic acid and palmitic acid showed less influences on I-FABP expression. However, in the present study, I-FABP mRNA level was not affected by α-linolenic acid, which suggested that I-FABP gene expression in C. livia was probably regulated by n-6 polyunsaturated fatty acids. In summary, this is the first report of identification and characterization of I-FABP in pigeon C. livia. Pigeon IFABP shared high identity with other avian I-FABP, and it belonged to the first subfamily of FABPs. Cytosolic fattyacid binding proteins signature and lipocalin domain were also detected on the amino acid sequence. Pigeon I-FABP gene expression showed a distinct development pattern which is different from that in mammals, and it also can be regulated by linoleic acid and arachidonic acid. Acknowledgments The authors thank all the members in the institute for their generous technical advice. This research was supported by National Natural Funds of China (No. 30871803 and No. 31072047). References Alvaro A, Rosales R, Masana L and Vallvé JC. 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