Philippine Journal of Science 148 (3): 433-439, September 2019 ISSN 0031 - 7683 Date Received: 22 February 2019

Comparative Expression Analysis of Small Intestine Nutrient Transporters Sodium/Glucose 1 (SGLT1) and Peptide Transporter 1 (PepT1) between Itik Pinas (Anas platyrhynchos L.) and Commercial Layer Chicken (Gallus gallus domesticus)

Alexandrinne M. Pinca, Herald Nygel F. Bautista, Christine B. Adiova, and Percival P. Sangel*

Institute of Animal Science, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Los Baños 4030 Laguna, Philippines

Itik Pinas (IP) is an improved breed of egg-type Philippine mallard duck (Anas platyrhynchos L.) that was developed to address the low egg production of the traditional mallard ducks. However, the improvement in genetic potential can only be realized if appropriate care and management – including feeding system – will be provided to the ducks. The energy and nutrient requirements of IP has yet to be established. Most IP raisers use commercial layer diets that are being formulated based on nutrient recommendations for chickens. However, differences in digestive physiology among poultry species might cause differences in their nutrient requirements. One way to better understand the digestive capacity and absorption efficiency of IP is to know the expression levels of nutrient transporter in their small intestine. Two nutrient transporters, specifically sodium/ glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), were analyzed from the small intestine of IP and were compared with those of layer chicken. Total RNA was independently isolated from the three main segments of the small intestine (i.e., duodenum, jejunum, and ileum) of IP-Kayumanggi and commercial layer chicken (CLC). The relative mRNA expression levels of the target genes from each of the intestinal segment in IP- Kayumanggi were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and were compared to the respective relative mRNA expression levels of the target genes in CLC. Results showed that SGLT1 has significantly higher relative mRNA expression levels in the three intestinal segments of IP-Kayumanggi compared to that in CLC. SGLT1 levels in IP were greatest in the ileum and jejunum. PepT1 relative mRNA expression levels in IP from the three intestinal segments, however, were shown to be comparable with that of CLC. Dissociation curve analysis showed a single peak, which validated the fidelity of the results. These novel findings suggest higher absorptive capacity of IP for monosaccharides, which may lead to higher energy value of feed ingredients for IP compared to CLC. Further studies must be conducted to determine the feeding value of feeds specific for IP.

Keywords: commercial layer chicken, Itik Pinas, SGLT1, PepT1, Pfaffl Method, real-time qPCR

INTRODUCTION an improved egg-type Philippine mallard duck (Anas platyrhynchos L.) developed through the joint effort of the IP is a product of continuous breeding and genetic Philippine Council for Agriculture, Aquatic, and Natural selection of the traditional native or Pateros duck popularly Resources Research and Development of the Department raised locally in the Philippines (Parungao 2017). It is of Science and Technology (DOST-PCAARRD) and the *Corresponding Author: [email protected] National Swine and Poultry Research and Development

433 Philippine Journal of Science Pinca et al.: Intestinal Nutrient Transporters Expression Vol. 148 No. 3, September 2019 in Itik Pinas vs. Commercial Layer Chicken

Center of the Bureau of Animal Industry (BAI-NSPRDC) number CAFS-2018-005. This study was conducted in to further boost the local duck egg production. However, the Advanced Animal Science Research and Training their energy and nutrient requirements are yet to be Laboratory of the Institute of Animal Science, College of established. Most IP raisers rely on commercially available Agriculture and Food Science (CAFS), UPLB. layer diets that were formulated based on chicken’s requirements. One way to better understand the digestive Management and Selection of Animals for Sampling capacity and absorption efficiency of the small intestine Six (6) IP-Kayumanggi and five (5) H&N CLC were of IP as it compared with CLC is to know the expression reared in individual metabolic cages. All animals levels of the different nutrient transporters. received restricted feeding computed based on their In the small intestine, nutrients are absorbed through a body weight, using the nutrient requirement designed variety of nutrient transporters or transport (Insel et for CLC. Water was offered ad libitum to all birds. al. 2012). These transporters are located mainly in the apical Everyday, IP-Kayumanggi were given 150 g of the border of intestinal in the mucosal epithelial commercially-formulated layer diet, while 110 g of the tissue, which aid in the transport of nutrients from the same diet was given to the CLC. Artificial light (20-W intestinal lumen into the cell during the digestion process. fluorescent bulb) was installed to provide the layers with 16 hours of required length of light exposure for efficient This study focused on two nutrient transporters – a egg production. After three to four weeks, three (3) IP- monosaccharide transporter, SGLT1, which transports Kayumanggi at 26 weeks of age and three (3) H&N CLC glucose and galactose through facilitated diffusion co- at 19 weeks of age were randomly selected, both on their transported by an electrochemical gradient (Harada and respective first week of uniform laying period. At this Inagaki 2010); and a peptide transporter, PepT1, which period, both the ducks and the chickens have already transports dietary amino acids across the brush border reached their sexually matured body weight. membrane as di- and tripeptides using an H+-dependent mechanism (Frazier et al. 2008). Collection and Preparation of Intestinal Tissue Samples Generally, amino acid transporters are expressed greater in Tissue samples from the three main intestinal segments the distal part of the small intestine than the proximal part, of the selected IP-Kayumanggi and H&N CLC were whereas the monosaccharide transporters are expressed collected (Figure 1). Specifically, 20 mm of duodenum, greater in the jejunum than the duodenum and ileum jejunum, and ileum tissue samples were measured and (Weintraut et al. 2015). Intestinal nutrient transporters obtained from the mid-duodenal loop, start of mesenteric are known to be adaptive, primarily to the dietary levels artery towards jejunum, and 50 mm from ileocecal of their substrates and the stage of development of the junction towards the ileum, respectively. Intestinal animal. For instance, higher expression of SGLT1 mRNA segments were then immediately rinsed with ice-cold 1X was seen to be related to the higher activity of the SGLT1 PBS (NaH2PO4, 1.47 mM; Na2HPO4, 8.09 mM; NaCl, transporter, while greater levels of dietary were 145 mM). About 30 mg of the rinsed segments were associated with greater expression of PepT1 mRNA and minced using a sterile blade and transferred into a sterile these mRNA levels varied with both dietary protein level Eppendorf tube. Aliquots were frozen and stored at –80 and developmental stage (Gilbert 2008). °C bio-freezer for subsequent analysis. Most of the commercially available duck layer feeds adopt the nutrient specifications of feed ingredients established for chickens. Likewise, majority of the commercially available layer diets are based on the nutrient recommendations for layer chickens, which may not optimally fit with the IP’s digestive potential. Thus, the aim of this study is to compare the mRNA expression levels of SGLT1 and PepT1 between IP and CLC through RT-qPCR analysis.

MATERIALS AND METHODS Figure 1. Gross morphology of the internal organs of a layer Experimental procedures were approved by the Animal chicken showing the three small intestinal segments Care and Use Committee of the University of the (A – duodenum, B – jejunum, C – ileum) where tissue Philippines Los Baños (UPLB) with assigned protocol samples for total RNA extraction were taken.

434 Philippine Journal of Science Pinca et al.: Intestinal Nutrient Transporters Expression Vol. 148 No. 3, September 2019 in Itik Pinas vs. Commercial Layer Chicken

Extraction and Isolation of Total RNA housekeeping genes were performed and quantified using Total RNA was extracted from the intestinal tissue samples the same procedure and RT-qPCR conditions used in (i.e., duodenum, jejunum, and ileum) using the Promega generating the standard curves. SV Total RNA Isolation System based on the protocol recommended by the manufacturer for small tissue samples Dissociation curve was generated using triplicates per (≤30 mg). The concentration of total RNA collected was with 10 ng/uL concentration and no template or quantified spectrophotometrically at 260/280 nm using negative control through GoTaq® 1-Step RT-qPCR Take3 Micro-Volume Plates and BioTek™ Epoch™ Reagent System (Promega Corporation, USA) with Microplate Spectrophotometer (Winooski, VT, USA) SYBR® Green as the detection dye. The dissociation and analyzed using Gen5™ 2.09 Take3 microplate data stage RT-qPCR conditions were 95 °C for 15 s, 60 °C for analysis software. Three (3) 30 μL aliquots of per tissue 1 min, and 95 °C for 15 s. A total reaction volume of 10 sample were put in a 1.5 mL Eppendorf tube each and uL per well was used for all reactions. stored again at –80 °C bio-freezer. Statistical Analysis Relative mRNA expression levels were determined using One-Step Real-time Quantitative Polymerase Chain Pfaffl method and were normalized with reference to Reaction (1-Step RT-qPCR) the housekeeping gene (GAPDH) expression level. The Quantitative analysis of the total RNA isolate was resulting values from three independent experiments measured for the number of molecules of RNA present were analyzed using SAS University Edition software. for each gene of interest per nanogram of total RNA Unpaired Student t-test with significance level of starting template using 1-step RT-qPCR. Specific primers 0.05 (95% confidence level) was conducted to detect for the housekeeping and target genes used for real-time differences on the expression levels of intestinal nutrient quantification were based on the primers designed by transporters between IP-Kayumanggi and H&N CLC. Gilbert et al. (2007) using Primer Express software (Applied Biosystems Real-Time PCR Systems). All oligonucleotides used for the study is summarized in Table 1. RESULTS AND DISCUSSION Five-point standard curves with total RNA concentrations of 50.0, 10.0, 2.0, 0.4, and 0.8 ng/μL (1:5 dilution series) Isolation of Total RNA for the target genes, SGLT1 and PepT1 – including the One (1) intestinal tissue aliquot was selected from each of housekeeping gene, GAPDH – were generated through the intestinal segments for the isolation and purification GoTaq® 1-Step RT-qPCR Reagent System performed on of total RNA, which was performed using the previously a 96-well plate Applied Biosystems StepOnePlus™ Real- described protocol for small tissue samples (≤30 mg). Time PCR System (Foster City, CA, USA). Each of the Overall, the concentrations of the total RNA isolated from points in the standard curve was carried out in triplicate. each of the three segments from the two animal samples A negative control for each gene was also placed which, that were quantified spectrophotometrically at 260/280 by definition, was expected to have no effect or to show nm can be classified as pure since all A260/280 values are an undetermined value. approximately equal to 2.0. This also suggests that the isolated RNA from the samples are relatively free from The following RT-qPCR conditions were used: 50 °C for proteins and inhibitors that may cause undesirable qPCR 5 min and 95 °C for 5 min during the holding stage; and results. 36 cycles of 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, and a final step of 73 °C for 10 min during the Standard Curve and Amplification Efficiency cycling stage. On the other hand, 10 ng/uL RNA samples For the standard curve, one (1) 30 μL aliquot was used as from the three intestinal segments of both species were a template to generate the equation of regression line for used as a template for the amplification of the target and standard curve for the housekeeping gene and the target housekeeping genes. The amplification of the target and gene with the concentrations and RT-qPCR conditions

Table 1. Forward and reverse primer sequences for SGLT1, PepT1, and GAPDH. Gene Forward sequence Reverse sequence SGLT1 GCCATGGCCAGGGCTTA CAATAACCTGATCTGTGCACCAGTA PepT1 CCCCTGAGGAGGATCACTGTTGGCAGTT CAAAAGAGCAGCAGCAACGA GAPDH GCCGTCCTCTCTGGCAAAG TGTAAACCATGTAGTTCA

435 Philippine Journal of Science Pinca et al.: Intestinal Nutrient Transporters Expression Vol. 148 No. 3, September 2019 in Itik Pinas vs. Commercial Layer Chicken described previously. In addition to that, the amplification efficiencies and percent efficiencies of the housekeeping gene – GAPDH – as well as the target genes SGLT1 and PepT1 were calculated using the slope of the regression line (Equation 1) in the standard curve:

(1)

Values of the equation of the line and the calculated amplification efficiency are shown in Table 2.

Table 2. Regression line and amplification efficiencies of GAPDH, PepT1, and SGLT1. % R2 Y- Amplification Efficiency GENE Slope (≥0.98) intercept efficiency E (90– 105%) GAPDH 0.999 –3.38 17.29 1.98 97.63 PepT1 0.992 –3.38 28.11 1.98 97.71 SGLT1 0.989 –3.27 24.05 2.02 102.21

Pfaffl Method and Relative mRNA Expression Levels The average Ct values obtained from all three (3) segments of chicken samples were used as the control values. ∆Ct were calculated separately from each gene wherein the average Ct from each gene in each segment of the IP- Kayumanggi was subtracted from their corresponding control values. Using the amplification efficiencies and ∆Ct values obtained, relative gene expression ratio – while accounting for differences in primer efficiencies – were determined using the Pfaffl method (Pfaffl 2004):

(2)

By default, gene expression ratio equal to 1 was used for all control values. Hence, values greater or less than 1 Figure 2. Expressions of PepT1 and SGLT1 in the three intestinal correspond to a fold increase or decrease in expression segments (A – duodenum, B – jejunum, C – ileum) of IP- Kayumanggi were assessed by RT-qPCR and the changes with respect to control. Furthermore, to test the significance are presented as a fold change relative to their expression of the differences observed, unpaired Student’s t-test was levels in H&N CLC used as controls. Both PepT1 and performed. All calculated data were summarized in Table 3. SGLT1 mRNA levels were normalized to GAPDH levels. Comparison of relative mRNA expression levels of PepT1 Significance was assessed and compared with the levels and SGLT1 in the duodenum, jejunum, and ileum were in the control using unpaired Student's t-test (*p < 0.05). shown in Figures 2A, 2B, and 2C, respectively. Error bars represent standard error from the mean from three independent experiments. IP-Kayumanggi exhibited an increase in relative mRNA expression levels of both genes (PepT1 and SGLT1) in the three (3) intestinal segments, which means that both genes be noted that SGLT1 mRNA expression of IP-Kayumanggi were upregulated in the duodenum, jejunum, and ileum was greatest at ileum and jejunum, where there is a with respect to their relative expression levels in H&N fifteen- and seven-fold increases observed, respectively. CLC, which was used as control in this study. However, These results suggest that IP’s intestinal segments can only the expression levels of SGLT1 in the duodenum (p = possibly absorb a much greater concentration of glucose 0.017), jejunum (p = 0.044), and ileum (p = 0.028), were and galactose than those of CLC. Higher absorption of found to be significantly different from the control. It can monosaccharides in IP may imply higher energy value of feed ingredients for IP compared to CLC. This finding

436 Philippine Journal of Science Pinca et al.: Intestinal Nutrient Transporters Expression Vol. 148 No. 3, September 2019 in Itik Pinas vs. Commercial Layer Chicken

Table 3. Relative gene expression ratio using Pfaffl method and T-test values of PepT1 and SGLT1 in the duodenum, jejunum, and ileum of experimental (IP-Kayumanggi) and control (H&N CLC) samples. Relative gene expression ratio came from three independent experiment (i.e., S1, S2, and S3). Each independent experiment was run in triplicates for both the experimental and control samples. Genes Ratio S1 Ratio S2 Ratio S3 Ave SD N SEM Ctrl 1 Ctrl 2 Ctrl 3 Ave SD N SE T-test Duodenum PepT1 0.0376 1.4355 4.3197 1.9309 2.1836 3 1.2607 1 1 1 1 0 3 0 0.5372 SGLT1 2.3324 3.0304 2.4561 2.6063 0.3725 3 0.2150 1 1 1 1 0 3 0 0.0175 Jejunum PepT1 1.3661 1.7484 0.7188 1.2777 0.5204 3 0.3005 1 1 1 1 0 3 0 0.4529 SGLT1 4.3984 8.4665 8.1366 7.0005 2.2595 3 1.3045 1 1 1 1 0 3 0 0.0442 Ileum PepT1 3.8467 1.4414 1.0574 2.1152 1.5118 3 0.8728 1 1 1 1 0 3 0 0.3296 SGLT1 14.0622 11.3442 19.5379 14.9814 4.1735 3 2.4096 1 1 1 1 0 3 0 0.0284 can have a positive impact on lowering feed cost. If the mechanism for the lack of substrate (Gilbert 2008, Gilbert specific energy values of feed ingredients for IP will be et al. 2010). However, either possible mechanism could used in formulating diets, resource use efficiency can be only be confirmed and validated by digestibility study, improved and feed cost may be reduced. which is highly recommended for future analysis. The absorption of carbohydrates in the intestine is vital Since incorporation of more protein in the diet is costly for maintaining energy in animals – which is influenced and can add to the increased nitrogen excretion in the by luminal digestion, apical membrane digestion, and environment, greater understanding on how the diet transport into the by SGLT1 (Sklan 2003, Gal- influences PepT1 mRNA expression levels of IP and Garber et al. 2000) – especially during the first week of how nutrient transporters respond to the diet can be used laying period wherein additional energy is needed for egg for the development of a least-cost feed formulation that production. The upregulation of SGLT1 especially in the adequately satisfies the animal’s requirement. jejunum can be due to interaction of intestinal segment and age wherein SGLT1 mRNA expression increases Dissociation Curve with age (Gilbert 2008). Likewise, Gilbert et al. (2007) Shown in Figure 3 are the melt curves or dissociation also showed a greater expression of SGLT1 mRNA in the curves for the three genes that were generated using the jejunum and ileum as compared to the duodenum of the qPCR concentrations and conditions that were previously chickens. Also, since jejunum and ileum are the major sites described. All of the three genes exhibited a single peak or of disaccharidase activity, high mRNA expression levels melting temperature (Tm), which means that the desired on these segments are highly expected. Moreover, several amplicon was detected. It also means that the primers are studies showed greater expression of SGLT1 at jejunum giving a specific PCR product and there is no non-specific than duodenum and ileum, suggesting that it is the primary amplification, thus increasing the fidelity of the results site of sugar assimilation in the chicken intestine (Gilbert generated from RT-qPCR. 2008, Gorboulev et al. 2012). Interestingly, in this study, SGLT1 mRNA was greatest in the ileum. For PepT1 mRNA, expression level for both poultry species were relatively the same, suggesting same CONCLUSION absorptive capacity of the small intestine for peptides. It This is the first attempt to study the nutrient transporters, can be observed that PepT1 mRNA expression level was PepT1 and SGLT1, in the three segments of the small greatest at duodenum and ileum even though a relatively intestine of IP via RT-qPCR in comparison with that of similar or comparable expression of PepT1 across the CLC. three segments can be observed. This is in agreement with previous studies in chickens, wherein PepT1 was It was found out that IP-Kayumanggi exhibits an expressed at greatest levels in the duodenum (Chen et upregulated or higher mRNA expression level for al. 2002, 2005). The upregulation of PepT1 may indicate SGLT1 compared to CLC. Specifically, SGLT1 mRNA two (2) reasons: 1) it is a mechanism to take advantage expression was greatest at ileum and jejunum, which was of the abundance of substrate, or 2) it is a compensatory expected since jejunum and ileum are the major sites of

437 Philippine Journal of Science Pinca et al.: Intestinal Nutrient Transporters Expression Vol. 148 No. 3, September 2019 in Itik Pinas vs. Commercial Layer Chicken

specific amplification and, thus, validating the fidelity of the results generated from RT-qPCR. While this study showed an overview of the molecular mechanism of nutrient transport and relative mRNA expression levels between IP and CLC, further analysis to validate results – such as digestibility studies and compositional analysis, along with RT-qPCR analysis of these and other nutrient transporters – is recommended.

ACKNOWLEDGMENT This work was supported by the DOST-PCAARRD funded project titled “Establishment of Nutrient Requirements of Improved Philippine Mallard Ducks” implemented by the Institute of Animal Science, CAFS, UPLB.

STATEMENT ON CONFLICT OF INTEREST All authors have no conflict of interest to declare.

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