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Expression of Small Intestinal Nutrient Transporters in Embryonic and Posthatch Turkeys
M. L. Weintraut*, S. Kim†, R. A. Dalloul*, and E. A. Wong*1
*Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061
† The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH25
9RG, UK
* Corresponding author:
Eric A. Wong, Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA
24061, Ph. 540-231-4737, FAX 540-231-3010, [email protected]
Running Head: NUTRIENT TRANSPORTERS IN TURKEYS
Section: Molecular and Cellular Biology
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ABSTRACT
Nutrients are absorbed in the small intestine through a variety of transporter proteins, which have
not been as well characterized in turkeys as in chickens. The objective of this study was to
profile the mRNA expression of amino acid and monosaccharide transporters in the small
intestine of male and female turkeys. Jejunum was collected during embryonic development
(E21, E24 and day of hatch) and duodenum, jejunum, and ileum were collected in a separate
experiment during posthatch development (Day of hatch, D7, D14, D21, D28). Real-time PCR
was used to determine expression of aminopeptidase N (APN), one peptide (PepT1), six amino
acid (ASCT1, bo,+AT, CAT1, EAAT3, LAT1, y+LAT2) and three monosaccharide (GLUT2,
GLUT5, SGLT1) transporters. Data were analyzed by ANOVA using JMP Pro 11.0. APN, bo,
+AT, PepT1, y+LAT2, GLUT5 and SGLT1 showed increased expression from E21 and E24 to
DOH. During posthatch, all genes except GLUT2 and SGLT1 were expressed greater in females
than males. GLUT2 was expressed the same in males as females and SGLT1 was expressed
greater in males than females. All basolateral membrane transporters were expressed greater
during early development then decreased with age; while the brush border membrane
transporters EAAT3, GLUT5 and SGLT1 showed increased expression later in development.
Because turkeys showed high level expression of the anionic amino acid transporter EAAT3, a
direct comparison of tissue-specific expression of EAAT3 between chicken and turkey was
conducted. The anionic amino acid transporter EAAT3 showed 6-fold greater expression in the
ileum of turkeys at D14 compared to chickens. This new knowledge can be used to not only
better formulate turkey diets to accommodate increased glutamate transport, but to also optimize
nutrition for both sexes.
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Key words: nutrient transporters, turkey, qPCR, EAAT3
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INTRODUCTION
The transition from the yolk to the intestinal lumen as the main source of nutrients for a chick
is well documented (Noy and Sklan, 1998; Moran, 2007). During late embryogenesis, the yolk
sac decreases in size and the small intestine becomes more developed as the embryo prepares to
absorb nutrients from the intestinal lumen. Compared to chicks and ducklings, turkey poults have
an under-developed gastrointestinal tract (Uni et al., 1999; Applegate et al., 2005). Thus,
understanding developmental changes in transporter expression in the intestine is important to
better utilize nutrients in feedstuffs in poultry.
Nutrient transporters belong to the solute carrier (SLC) gene family, which contains 395
genes organized into 52 families in the human genome (Hediger et al., 2013). Transporters
present in the intestinal enterocyte are located at both the brush border membrane for transport of
nutrients from the intestinal lumen into the cell and at the basolateral membrane for transport of
nutrients into or out of the bloodstream. Amino acids are transported either in a free form
through a variety of neutral, anionic, or cationic amino acid transporters or as short peptides
through peptide transporters. Examples of these transporters include the alanine, serine, cysteine
and threonine transporter 1(ASCT1) and excitatory amino acid transporter 3 (EAAT3), which
transport mainly neutral and anionic amino acids, respectively and are members of the SLC1
family (Kanai et al., 2013). Solute carrier family 7 (SLC7) members include bo,+AT, the cationic
(CAT1), large amino acid (LAT1), and y+L amino acid (y+LAT2) transporters (Fotiadis et al.,
2013). The peptide transporter PepT1 is a member of the SLC15 family, which transports small
oligo-peptides (Smith et al., 2013). Monosaccharide transporters are also members of the SLC
gene family. The sodium-glucose co-transporter SGLT1 belongs to the SLC5 family and
transports glucose and galactose (Wright, 2013). Members of the SLC2 family include the
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facilitated sugar transporters GLUT5, which transports fructose, and GLUT2, which transports
glucose, galactose, mannose and fructose (Mueckler and Thorens, 2013).
The developmental expression of nutrient transporters has been profiled during the
embryonic and posthatch periods in chickens (Gilbert et al., 2007; Li et al., 2008; Zeng et al.,
2011; Li et al., 2012; Speier et al., 2012; Zwarycz and Wong, 2013; Miska et al., 2014, 2015)
and pigeons (Dong et al., 2012; Chen et al., 2015). Generally, amino acid transporters are
expressed greater in the distal than the proximal part of the small intestine, whereas the
monosaccharide transporters are expressed greater in the jejunum than the duodenum and ileum.
Many brush border membrane transporters showed increased expression with developmental
age, whereas basolateral membrane transporters showed decreased expression. In turkeys, the
expression patterns of a peptide (PepT1) and monosaccharide (SGLT4) transporter have been
examined in the duodenum of embryos from embryonic day 20 to day of hatch (DOH) but not
posthatch (de Olivera et al., 2009). The objective of this study was to provide a comprehensive
profile of the expression of a digestive enzyme, a peptide transporter, six amino acid, and three
monosaccharide transporters from late embryogenesis to 4 weeks posthatch in turkeys.
MATERIALS AND METHODS
Birds and Tissue Collection
All animal procedures were approved by the Institutional Animal Care and Use Committee at
Virginia Tech. For the prehatch experiment, 100 fertile commercial turkey eggs were obtained
from AgForte (Harrisonburg, VA). Eggs were incubated at 37.5 C and 55% humidity with
turning every 45 minutes. Most turkeys hatched on d 27 of incubation. On embryonic d 21 (E21),
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E24 and DOH, jejunum was collected from 6 males and 6 females, which were visually sexed.
All samples were stored at -80C until RNA extraction.
For the posthatch experiment, 50 male and 50 female DOH turkey poults (AgForte) were
housed separately on large floor pens with fresh wood shavings and provided with starter
commercial poult feed and water ad libitum (Kim et al., unpublished). On DOH and d 7, 14, 21
and 28 posthatch, duodenum, jejunum, and ileum were collected from 6 males and 6 females and
stored in RNAlater (Life Technologies, Grand Island, NY).
The sex of the birds was further verified using PCR. Genomic DNA was extracted using the
manufacturer’s protocol for the Quick-gDNA kit (Zymo Research, Irvine, CA) and quantified
using a Nanodrop (Thermo Scientific, Wilmington, DE). Two primers for the W Chromosome
(Forward: 5’-GGGTGTAACATGAGAAGAAC-3’ and Reverse: 5’-
GCACAGATGGAGACAAAAGC-3’) (Kalina et al., 2012) and two primers for the autosomal
gene PepT1 (Forward: 5’-TTGTCTCCCTGTCCATTGTCTATAC-3’ and Reverse: 5’-
GTTCTTCAAACTGATCCCCACCAAA-3’) were used for PCR sexing. PCR reactions
contained 12.5 µL of Accustart II PCR SuperMix (Quanta Biosciences, Gaithersburg, MD), 200
nM of each of the four primers, 1 ng of DNA and diethylpyrocarbonate treated-H2O to make a
final volume of 25 µL. The PCR conditions were: 94 C for 10 min followed by 30 cycles of 94
C for 30 s, 54 C for 20 s, and 72 C for 40 s. The PCR products were separated on a 1% agarose
gel. Male turkeys lacking the W chromosome exhibited only the PepT1 band of 384 bp. Female
turkeys exhibited two bands of 384 bp and 565 bp corresponding to PepT1 and the W
chromosome, respectively.
RNA Extraction and Relative qPCR for Turkey Transporters
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Total RNA was extracted from the embryonic (E21, E24) and DOH jejunal samples as per
the Direct-zol RNA Miniprep (Zymo Research) protocol and from the posthatch samples
(DOH, D7, D14, D21, and D28) using RNeasy Mini Spin Columns (Qiagen, Valencia, CA) with
the use of the QIAcube (Qiagen) according to the manufacturer’s instructions. The RNA
samples were quantified using a Nanodrop 1000 (Thermo Scientific) and then diluted to 200
ng/uL. The genes analyzed included aminopeptidase N (APN), the neutral (ASCT1, LAT1,
y+LAT2), Na+-independent (bo,+AT), anionic (EAAT3), and cationic (CAT1) amino acid
transporters, the peptide transporter PepT1 and the facilitated (GLUT2 and GLUT5) and Na+-
dependent (SGLT1) monosaccharide transporters (Table 1). cDNA was synthesized from 2 µg of
total RNA using the High Capacity Reverse Transcription cDNA kit (Applied Biosystems,
Foster City, CA). Synthesized cDNA was diluted 1:30 with nuclease-free water and 1 µL of
diluted cDNA was used in each qPCR reaction, which contained 5 µL of Fast SYBR Green
Mastermix (Applied Biosystems), 500 nM of both forward and reverse primers, and nuclease-
free water to make a final volume of 10 µL. The samples were run in a 7500 Fast Real-time PCR
instrument (Applied Biosystems) using the manufacturer’s default program (95°C for 20 s; 40
cycles of 95°C for 3 s and 60°C for 30 s). The primers for each of the 11 genes plus 18S rRNA
are listed in Table 1.
Fold change was calculated using the Ct method (Livak and Schmittgen, 2001).
Expression of rRNA served as the reference gene to calculate Ct. For the embryonic samples,
the average Ct of the jejunum of males at DOH was used as the calibrator to calculate Ct.
For the posthatch samples, the average Ct of the duodenum of males at DOH was used as the
calibrator, because expression was generally lowest in the duodenum. Statistical analysis was
performed using JMP Pro 11.0 software. All outliers were removed using Grubb’s test for
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outliers. Posthatch tissues were analyzed separately from embryonic tissues using ANOVA with
age, sex, and tissue (5 x 2 x 3 factorial) as the main effects and an =0.05 level of significance.
The embryonic samples were analyzed in a similar way with age and sex (3 x 2 factorial) as the
main effects. Tukey’s test was performed on all of the significant interactions.
Relative Quantification of Chicken and Turkey EAAT3
The chicken samples used for this experiment were Aviagen line A chickens used in a
previous study (Gilbert et al., 2007). Male chicken and male turkey duodenum, jejunum, and
ileum samples from DOH, D7, and D14 were compared (n=4). Total RNA was extracted as per
the Direct-zol RNA Miniprep (Zymo Research). cDNA was synthesized from 2 µg of total
RNA using the High Capacity Reverse Transcription cDNA kit (Applied Biosystems). One µL
of the diluted cDNA (1:30) was added to a 10 µL PCR reaction containing 5 µL of SYBR green
and 500 nM of both forward and reverse primers that anneal to the cDNA of both species (Table
1). The chicken and turkey samples were compared using the ΔΔCt method. GAPDH was the
reference gene and the calibrator was turkey duodenum at DOH. Results were analyzed with
JMP11.0 software using ANOVA with age, tissue and species (3 x 3 x 2 factorial) as the main
effects and an =0.05 level of significance. Tukey’s test was performed on all of the significant
interactions.
RESULTS
The expression of one aminopeptidase and 10 nutrient transporters was assayed in two
separate experiments. One experiment examined embryonic expression in the jejunum from E21
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until day of hatch. The second experiment examined posthatch expression in duodenum,
jejunum, and ileum on DOH, D7, D14, D21, and D28.
The expression of genes during embryogenesis is shown in Table 2, with separate rows
for main effects of sex and age and the sex x age interaction. For all genes examined, there were
no differences between males and females. The mRNA for genes whose proteins are located at
the brush border membrane showed age-dependent changes. Expression of APN, the amino acid
transporter bo,+AT, peptide transporter PepT1 and monosaccharide transporters SGLT1 and
GLUT5 was increased on DOH compared to both E21 and E24. There was a sex x age
interaction for EAAT3 (Figure 1), where expression in male turkeys was greater than female
turkeys at E21 but the same at E24 and DOH. Ew
The expression of genes posthatch is shown in Table 3, with separate rows for main effects
of sex, tissue and age and their interactions. APN and the transporters showed sex-, tissue-, and
development-specific patterns of expression. Nine genes (APN, bo,+AT, EAAT3, PepT1, GLUT5,
ASCT1, CAT1, LAT1, and y+LAT2) showed greater expression in females than males. SGLT1
was expressed greater in males than females, while GLUT2 was the same for both males and
females. APN and many of the amino acid transporters showed greater expression in the distal
than the proximal segment of the small intestine. APN and EAAT3 were expressed greater in
the ileum than the duodenum and jejunum and bo,+AT was expressed greater in the ileum than the
jejunum. LAT1 showed greater expression in the jejunum and ileum than the duodenum and
y+LAT2 showed greater expression in the ileum than the duodenum. In contrast, the
monosaccharide transporters showed a different pattern of expression. GLUT5 and GLUT2 were
expressed the greatest in the duodenum and jejunum, respectively and SGLT1 was expressed
greater in the jejunum than the duodenum. The brush border membrane transporters (EAAT3,
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GLUT5 and SGLT1) showed increased expression after DOH, while the basolateral membrane
transporters (CAT1, y+LAT2, and GLUT2) showed decreased expression after DOH.
Six genes showed 2-way interactions between sex, age, or tissue. The fructose transporter
GLUT5 showed a sex x tissue interaction, with females expressing greater GLUT5 mRNA than
males in the duodenum and jejunum, but not the ileum (Figure 2A). There was a sex x age
interaction with females expressing greater GLUT5 than males at D21 (Figure 2B); and a tissue
x age interaction with GLUT5 expression greater in the duodenum than the jejunum and ileum at
D7 (Figure 2C). The large, neutral amino acid transporter LAT1 displayed a sex x age
interaction with females expressing greater LAT1 than males at D7 (Figure 3A); and a tissue x
age interaction with greater expression of LAT1 in the jejunum and ileum than the duodenum at
D7 (Figure 3B).
Four other genes showed tissue x age interactions. There was greater expression in the ileum
than the duodenum and jejunum for both APN at D28 (Figure 4A) and the anionic amino acid
transporter EAAT3 at D14, D21, and D28 (Figure 4B). For the monosaccharide transporters,
there was greater expression of SGLT1 in the jejunum than the duodenum at D28 (Figure 4C),
while there was greater expression of GLUT2 in the jejunum than ileum at DOH (Figure 4D).
Because EAAT3 appeared to be expressed at greater levels in turkeys than chickens, a direct
comparison between turkey and chicken EAAT3 mRNA abundance was conducted using
primers that amplified EAAT3 from both species (Figure 5). Overall there was approximately 2-
fold greater expression of EAAT3 in turkeys than chickens (P<0.001, data not shown). In
turkeys there was greater expression of EAAT3 mRNA in the ileum than the duodenum or
jejunum at DOH, D7 and D14, with a greater than 1000-fold increase in the ileum compared to
the duodenum at D14. In contrast, there was no difference between intestinal segments in the
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chicken. In the turkey ileum, there was greater than 5-fold increase from DOH and D7 to D14;
whereas in the chicken ileum there was no change from DOH to D14. At D14 in the ileum,
turkey EAAT3 was 6-fold greater than chicken EAAT3.
DISCUSSION
As the embryo prepares to hatch, the seroamniotic connection ruptures allowing oral
consumption of the albumin. While digestion at this stage is still limited due to antitrypsin
factors, some absorption is possible while in transit through the duodenum and jejunum (Moran,
2007). In general, expression of brush border membrane transporters increases from late
embryogenesis towards hatch, while basolateral transporters decrease from late embryogenesis
towards hatch. At the brush border membrane, expression of many amino acid (e.g., EAAT3, bo,
+AT, BoAT), peptide (e.g., PepT1), and monosaccharide (e.g., SGLT1, GLUT2, GLUT5)
transporters as well as the digestive enzymes sucrase isomaltase (SI) and APN increase during
embryogenesis in chickens (Gilbert et al., 2007; Li et al., 2008; Zeng et al., 2011; Speier et al.,
2012; Li et al., 2013; Miska et al., 2014) and pigeons (Dong et al., 2012; Chen et al., 2015). In
turkeys, there were similar increasing expression profiles towards hatch for APN, PepT1, bo,+AT,
GLUT5 and SGLT1 in this study and PepT1, SI and SGLT4 in the microarray study of de
Olivera et al. (2009). In chickens and pigeons, there was generally a decrease in mRNA
abundance of the basolateral membrane transporters CAT1, CAT2 and LAT1, and an increase in
y+LAT2 mRNA, although not all studies showed these expression profiles (Gilbert et al., 2007;
Li et al., 2008; Zeng et al., 2011; Speier et al., 2012; Miska et al., 2014; Chen et al., 2015). The
current study with turkeys showed decreased expression of ASCT1 and CAT1. The basolateral
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monosaccharide transporter GLUT2 was upregulated in turkeys similar to chickens and pigeons
(Gilbert et al., 2007; Dong et al., 2012; Li et al., 2015).
The general pattern of upregulation of mRNA for transporters at the brush border membrane
and downregulation at the basolateral membrane can be explained by the source of the nutrient
supply. During embryogenesis the avian embryo is dependent upon the yolk for nutrients. The
expression of transporters in the yolk sac (Yadgary et al., 2011; Dong et al., 2012; Speier et al.,
2012) mediates transport of nutrients from the yolk into the embryonic blood system, which are
carried to the intestinal enterocytes and are taken up by basolateral transporters. Prior to hatch as
the yolk is being depleted, the avian embryo begins to swallow amniotic fluid, which enters the
intestine as a source of nutrients. Upregulation of the brush border membrane transporters
allows uptake of these nutrients into the enterocyte. However, not all basolateral transporters are
downregulated. The monosaccharide transporter GLUT2, which is mainly located at the
basolateral membrane, was increased in turkey, chicken and pigeon. This upregulated GLUT2
expression would facilitate the export of monosaccharides, which have been taken up by the
enterocyte, into the blood to be delivered to tissues and organs of the newly hatched bird.
Posthatch tissue- and development-specific expression patterns for the nutrient
transporters in turkeys is comparable to that reported for chickens and pigeons (Gilbert et al.,
2007; Li et al., 2008; Dong et al., 2012; Miska et al., 2015). In turkeys, expression of APN and
many of the amino acid transporters (EAAT3, LAT1, y+LAT2) was greater in the ileum than the
duodenum. In contrast, expression of the monosaccharide transporters (SGLT1 and GLUT2)
was greater in the jejunum than the ileum. These patterns of expression are similar to those seen
in chickens (Gilbert et al, 2007; Miska et al., 2015) and pigeons (Dong et al., 2012). The
digestive enzyme APN and the brush border membrane transporters EAAT3, PepT1, SGLT1,
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and GLUT5 showed increased expression after hatch in chickens (Gilbert et al., 2007; Li et al.,
2008; Miska et al., 2015) and pigeons (Dong et al., 2012). In turkeys only EAAT3, SGLT1, and
GLUT5 showed increased expression after hatch, whereas APN and PepT1 were unchanged. At
the basolateral membrane, CAT1 was decreased in both turkeys and chickens (Gilbert et al.,
2007). In contrast, GLUT2 was increased in chickens and pigeons but decreased in turkeys
(Gilbert et al., 2007; Li et al., 2008; Dong et al., 2012).
At the brush border membrane, SGLT1 is the main transporter of glucose, while GLUT5
is a fructose transporter; while at the basolateral membrane, GLUT2 transports a variety of
monosaccharides (Mueckler and Thorens, 2013; Wright, 2013). Upregulation of SGLT1 would
enhance the uptake of glucose into the intestinal epithelial cells, which could then be exported
out of the cell into the blood for transport to tissues and organs by upregulated GLUT2
expression. The upregulation of GLUT5 is likely an innate response posthatch that prepares
birds to consume a diet of fruits containing fructose. Interestingly, this would no longer be
necessary for birds consuming a commercial corn-soybean based diet, but the gene induction is
maintained. Our results show that GLUT2 expression in turkeys was greater in females than
males at d21 (sex x age interaction) and in the duodenum and jejunum compared to the ileum
(sex x tissue interaction). These results suggest that female turkeys may have had a greater
capacity to absorb fructose from a diet of fruits and berries than males.
There is limited research concerning sexual dimorphism of nutrient transporters in
Galliformes. Prior to hatch, there was no difference in expression of any nutrient transporter
examined in male and female turkeys. In contrast, during embryogenesis (E9 to DOH) in
Wenshi Yellow-Feathered chickens, expression of CAT1, CAT4, and LAT4 was greater in males
than females, while expression of y+LAT2 was greater in females then males (Zeng et al., 2011).
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During posthatch, expression of the digestive enzyme APN and eight transporters (ASCT1,
b0,+AT, CAT1, EAAT3, LAT1, PepT1, y+LAT2 and GLUT5) was greater in female turkeys than
males. For GLUT5 and LAT1, this difference is likely due to the sex x age interaction where
female turkeys express greater GLUT5 at D21 and greater LAT1 at D7 than male trukeys. These
results are noteworthy as the official nutrient requirements for both male and female growing
turkeys are the same (National Research Council, 1994) but our data suggest that male and
female turkeys may assimilate nutrients differently. It is possible that females have a greater
need for amino acids and monosaccharides than males or that females utilize feed less
efficiently. In chickens, differences between males and females during posthatch have not been
reported, since most studies have focused on gene expression in males only.
The most striking difference between turkeys and chickens is the marked increase in EAAT3
expression on D14 in turkeys that has not been observed in chickens. EAAT3 transports anionic
amino acids such as glutamate, which is a major metabolic substrate in the enterocyte (Brosnan
and Brosnan, 2013). When chicken and turkey ileal samples from D14 were analyzed using the
same primers, EAAT3 expression was 6-fold greater in turkey than chicken. This difference
suggests that turkeys have an increased absorption capacity for glutamate in the ileum. In
turkeys, glutamate is the most digestible amino acid in birds fed a soybean meal diet, whereas
chickens and ducks do not demonstrate this increased digestibility for glutamate (Kluth and
Rodehutscord, 2006).
With the increasing understanding of genetics, nutrition, and metabolism of commercially
grown poultry, there is an opportunity to better formulate turkey diets to meet the need of both
males and females, and to possibly improve feed conversion and body weight gain with
knowledge emerging about amino acid, oligo-peptide, and monosaccharide transporters.
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ACKNOWLEDGMENTS
This work was supported in part by USDA National Institute of Food and Agriculture multistate
projects (EAW and RAD) and an Animal Genome Program Grant #2010-65205-20412 (RAD).
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characterization of aminopeptidase N from chicken intestine with potential application in
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Miska, K. B., R. H. Fetterer, and E. A. Wong. 2014. The mRNA expression of amino acid
transporters, aminopeptidase N, and the di- and tri-peptide transporter PepT1 in the embryo of
the domesticated chicken (Gallus gallus) shows developmental regulation. Poult. Sci. 93:2262-
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Miska, K. B., R. H. Fetterer, and E. A. Wong. 2015. mRNA expression of amino acid
transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1 in the intestine and
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Figure Captions
Figure 1. Interaction between age and sex for embryonic expression of EAAT3
Relative quantification of gene expression was determined using real time PCR with 18S
ribosomal RNA as the reference gene. Bars represent the means ± SEM (n=6) for relative
abundance of EAAT3 mRNA for male and female turkeys at embryonic days 21 (E21) and 24
(E24) and day of hatch (DOH) (n=6). Bars with a different letter (a-b) are significantly different
(P<0.05) when analyzed with Tukey’s test.
Figure 2. Interaction between age, sex and tissue for posthatch expression of GLUT5.
Relative quantification of gene expression was determined using real time PCR with 18S
ribosomal RNA as the reference gene. Bars represent the means ± SEM for relative abundance
of GLUT5 mRNA. A. Sex x tissue interaction for male and female turkeys in the duodenum
(Duo), jejunum (Jej) and ileum (Ile) (n=30). B. Sex x age interaction for male and female turkeys
on day of hatch (DOH), D7, D14, D21, and D28 (n=18). C. Tissue x age interaction in the
duodenum (duo), jejunum (jej) and ileum (ile) on DOH, D7, D14, D21, and D28 posthatch
(n=12). Bars with a different letter (a-e) are significantly different (P<0.05) when analyzed with
Tukey’s test.
Figure 3. Interaction between age, sex and tissue for posthatch expression of LAT1.
Relative quantification of gene expression was determined using real time PCR with 18S
ribosomal RNA as the reference gene. Bars represent the means ± SEM for relative abundance
of LAT1 mRNA. A. Sex x age interaction for male and female turkeys on day of hatch (DOH),
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D7, D14, D21, and D28 (n=18). B. Tissue x age interaction in the duodenum (duo), jejunum
(jej) and ileum (ile) on DOH, D7, D14, D21, and D28 posthatch (n=12). Bars with a different
letter (a-c) are significantly different (P<0.05) when analyzed with Tukey’s test.
Figure 4. Interaction between age and tissue for posthatch expression of APN, EAAT3,
SGLT1 and GLUT2. Relative quantification of gene expression was determined using real time
PCR with 18S ribosomal RNA as the reference gene. Bars represent the means ± SEM for
relative abundance of A) APN, B) EAAT3, C) SGLT1, and D) GLUT2 mRNA showing tissue x
age interaction in the duodenum (Duo), jejunum (Jej) and ileum (Ile) on DOH, D7, D14, D21,
and D28 posthatch (n=12). Bars with a different letter (a-c) are significantly different (P<0.05)
when analyzed with Tukey’s test.
Figure 5. Relative quantification of EAAT3 in chicken and turkey. Relative quantification of
gene expression was determined using real time PCR with GAPDH as the reference gene. Bars
represent the means ± SEM (n=4) for relative abundance of turkey and chicken EAAT3 mRNA
in the duodenum (Duo), jejunum (Jej) and ileum (Ile) at day of hatch (DOH) and D7 and D14
posthatch. Bars for each gene with a different letter (a-e) are significantly different (P<0.05)
when analyzed with Tukey’s test.
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Table 1. Real-time PCR primers for turkey nutrient transporters, aminopeptidase N and reference genesSLC family Gene Accession # Function PCR Primers (5’→3’); forward/reverse8 SLC1A11 EAAT3 XM_010725627 Glu, Asp transporter CCCAACGCTTGGACTTGTCA/
CAGCTGGCAGGCCAACASLC1A41 ASCT1 XM_010706509 Ala, Ser, Cys, Thr transporter AGGAAACGTCGCCTTTGGT/
GCTGTACTGGTTGTGTTGGAGACTSLC2A22 GLUT2 XM_010716927 Glucose, galactose, fructose,
mannose transporter TTTTCGAGAGAGCCGGTGTT/ TCACCACTCCAACGCCAAT
SLC2A52 GLUT5 XM_010722761 Fructose transporter CAACTCTCCAGCCCCCTACA/ GGAGACTCCGTGCCTGTTGA
SLC5A13 SGLT1 XM_003211023 Glucose and galactose transporter
GGGACAGTAGGTGGATTCTTTCTG/ CACCAATCGGCCACCAA
SLC7A14 CAT1 XM_003203401 Cationic amino acid transporter
TGGCCTTTCTCTTCGACTTGA/ CCAGGAGGGTCCCAATAGACA
SLC7A54 LAT1 NM_001030579 Large, neutral amino acid exchanger
AAGGCCCATCAAGGTGAACA/ AACAAGCAAGCCAGGATGAAG
SLC7A64 y+LAT2 XM_010717824 Cationic/ large neutral amino acid exchanger
TCTGCCTTGTTCTCTTATTCTGGTT/ TGGGTTTTTGATCTCCTCAGTCA
SLC7A94 bo,+AT XM_010717754 Cationic and large amino acid exchanger
TCCTTACCTTATGGAGGCCTTTG/ GCAGGCTTGCCCAAGAAAA
SLC15A15 PepT1 NM_001303166 Di- and tri- peptide transporter
TTTGACCAGCAGGGATCGA/ CAAAGTCCCCATCCATTGTTG
APN6 XM_003209631 N-terminal peptidase TGCGGGTGCGATGGA/ CGTTGTCATAGAGCAGCGAGTT
rRNA DQ018752.1 18S ribosomal RNA CCGTCGTAGTTCCGACCATAA/GCGGGTCATGGGAATAACG
SLC1A1 EAAT37 XM_010725627 (T)XM_424930.4 (C)
Glu, Asp transporter AATGCACTGAATGAAGCTACAATGA/CCAGCAATTAAAAACACAATACCAA
GAPDH7 NM_010718027.1 (T)NM_204305.1 (C)
Glyceraldehyde phosphate dehydrogenase
GCCGTCCTCTCTGGCAAAG/TGTAAACCATGTAGTTCA
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1 Kanai et al., 2013; 2 Mueckler and Thorens; 3 Wright, 2013; 4 Fotiadis et al., 2013; 5 Smith et al., 2013; 6 Mane et al., 20107 Primers that amplify both turkey and chicken EAAT3 and GAPDH. T (turkey), C (chicken)8 Primers designed by Primer Express software v3.0 (Applied Biosystems, Foster City, CA)
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Table 2. Embryonic turkey nutrient transporter gene expressionGene: APN bo,+AT EAAT3 PepT1 GLUT5 SGLT1 ASCT1 CAT1 LAT1 y+LAT2 GLUT2Attribute: Brush Border BasolateralSex
Male 0.49 0.71 2.14 0.37 0.63 0.69 1.55 2.42 1.32 0.66 0.93Female 0.49 0.75 1.43 0.62 0.80 0.71 1.15 1.95 1.22 0.63 1.01
SEM 0.06 0.09 0.32 0.15 0.17 0.09 0.24 0.26 0.14 0.07 0.12p-value 0.99 0.73 0.12 0.24 0.49 0.84 0.25 0.21 0.61 0.75 0.64
AgeE21 0.19b 0.45b 2.18 0.04b 0.26b 0.21b 1.96a 3.04a 1.32 0.45b 0.91ab
E24 0.18b 0.53b 1.68 0.06b 0.44b 0.21b 1.34ab 2.33a 1.33 0.35b 0.71b
DOH 1.08a 1.22a 1.50 1.16a 1.44a 1.68a 0.77b 1.19b 1.16 1.12a 1.28a
SEM 0.08 0.11 0.39 0.15 0.20 0.11 0.48 0.32 0.17 0.08 0.15p-value <0.0001 <0.0001 0.43 <0.0001 0.0005 <0.0001 0.029 0.0010 0.69 <0.0001 0.04
Interaction1
S x A 0.83 0.17 0.03 0.29 0.65 0.84 0.35 0.27 0.39 0.18 0.33a-b within a column indicates significant difference (P<0.05)1 For the interactions, S and A represent the main effects of sex and age, respectively
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450451452453454455456457458459460461462463464465
Table 3. Posthatch turkey nutrient transporter gene expression
Gene: APN bo,+AT EAAT3 PepT1 GLUT
5SGLT
1ASCT
1 CAT1 LAT1 y+LAT2
GLUT2
Attribute: Brush Border BasolateralSex
Male 3.70b 1.01b 36.02b 1.13b 2.80b 3.17a 0.98b 1.22b 1.15b 1.36b 1.58Female 6.11a 1.54a 58.28a 2.16a 4.34a 1.90b 1.82a 3.23a 2.02a 2.02a 1.91
SEM 0.42 0.12 4.63 0.11 0.29 0.24 0.15 0.42 0.15 0.11 0.19
p-value <0.0001
0.0025 0.0009 <0.000
1 0.0002 0.0003 0.0001 0.0008 <0.0001 <0.000
1 0.23
TissueD 3.43b 1.26ab 4.14b 1.78 5.72a 1.89b 1.24 1.85 0.89b 1.45b 1.85b
J 3.83b 0.94b 22.84b 1.73 3.34b 3.26a 1.61 2.27 1.66a 1.60ab 2.76a
I 7.45a 1.63a 114.48a 1.44 1.60c 2.45ab 1.35 2.56 2.23a 2.01a 0.63c
SEM 0.52 0.15 5.67 0.14 0.35 0.30 0.19 0.51 0.18 0.14 0.24
p-value <0.0001 0.010 <0.0001 0.16 <0.000
1 0.010 0.33 0.61 <0.0001 0.012 <0.0001
AgeDOH 3.52 1.45 33.46bc 1.80 1.73b 1.26b 2.10a 4.98a 1.94b 2.31a 2.56a
D7 5.98 1.08 37.60c 1.53 4.09a 2.03ab 1.55ab 2.14b 2.95a 1.51b 0.88b
D14 4.95 1.20 67.20a 1.56 4.34a 3.32a 1.01b 1.39b 1.19bc 1.76ab 1.83ab
D21 4.51 1.30 51.06abc 1.73 4.33a 3.18a 1.21ab 1.30b 0.80c 1.33b 1.83ab
D28 5.55 1.35 56.42ab 1.63 3.37ab 2.87a 1.13b 1.32b 1.06bc 1.52b 1.62ab
SEM 0.67 0.19 7.31 0.18 0.46 0.39 0.24 0.66 0.23 0.17 0.30
p-value 0.085 0.70 0.0008 0.80 0.0002 0.0008 0.011 0.0002
<0.0001 0.0011 0.0041
Interaction1
SxT 0.054 0.56 0.20 0.60 0.012 0.072 0.71 0.73 0.061 0.52 0.68
TxA 0.024 0.14 0.0002 0.14 <0.0001 0.049 0.36 0.94 0.044 0.17 0.042
SxA 0.63 0.11 0.77 0.49 0.0499 0.22 0.77 0.14 0.0498 0.70 0.46
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SxTxA 0.26 0.95 0.81 0.23 0.21 0.38 0.24 0.10 0.83 0.92 0.67a-c within a column indicates significant difference (P<0.05)1 For the interactions, S, T and A represent the main effects of sex, tissue, and age, respectively
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E21 E24 DOH0
0.5
1
1.5
2
2.5
3
3.5
a
ab
bb b
ab
male female
Age
Rel
ativ
e m
RN
A
abu
nd
ance
of E
AA
T3
Figure 1. Interaction between age and sex for embryonic expression of EAAT3
471472
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475
A. GLUT5 (Sex x Tissue) B. GLUT5 (Sex x Age)
Duo. Jej. Ile.0
1
2
3
4
5
6
7
8
b
cc
a
b
c
Male Female
Age
Fold
Ch
ange
DOH D7 D14 D21 D28
0
1
2
3
4
5
6
7
c
ab ab
bcbcbc
abab
a
ab
Male Female
Age
Fold
Ch
ange
C. GLUT5 (Tissue x Age)
DOH D7 D14 D21 D280
2
4
6
8
10
12
cde
a
bcab
bcde
cde de
bcdbcde
bcd
ede
cdecde cde
Duo. Jej. Ile.
Age
Fold
Ch
ange
Figure 2. Interaction between age, sex and tissue for posthatch expression of GLUT5.
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479480
A. LAT1 (Tissue x Age) B. LAT1 (Sex x Age)
DOH D7 D14 D21 D280
0.51
1.52
2.53
3.54
4.5
bcbc
bcc c
b
a
bcbc
bc
Male Female
Age
Fold
Ch
ange
DOH D7 D14 D21 D280
0.51
1.52
2.53
3.54
4.5
cc
c c c
bc
ab
cc c
abc
a
bcc
bc
Duo. Jej. Ile.
Age
Fold
Ch
ange
Figure 3. Interaction between age, sex and tissue for posthatch expression of LAT1.
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A. APN (Tissue x Age) B. EAAT3 (Tissue x Age)
DOH D7 D14 D21 D280
2
4
6
8
10
12
c
abc
bc bc bcabc
bc abcbc
bcabc
abcab ab
aDuo. Jej. Ile.
Age
Fold
Ch
ange
DOH D7 D14 D21 D280
20406080
100120140160180
c c c c cbc bc
bcbc bc
bbc
a
a aDuo. Jej. Ile.
Age
Fold
Ch
ange
C. SGLT1 (Tissue x Age) D. GLUT2 (Tissue x Age)
DOH D7 D14 D21 D280
1
2
3
4
5
6
b
ab abab
bb b
abab
a
bb
abab
ab
Duo. Jej. Ile.
Age
Fold
Ch
ange
DOH D7 D14 D21 D28
0
1
2
3
4
5
6
abcbc bc bc bc
a
bc
bc ababc
bcc
bcbc bc
Duo. Jej. Ile.
Age
Fold
Ch
ange
Figure 4. Interaction between age and tissue for posthatch expression of APN, EAAT3,
SGLT1 and GLUT2.
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DOH D7 D14 DOH D7 D14Chicken Turkey
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
de cdecde
e e e
bcde cde bcdee de cde
bcdebc bcd
b bc
aDuo. Jej.
Ile.
Age and Species
Rel
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RN
A a
bun
dan
ce o
f EA
AT
3
Figure 5. Relative quantification of EAAT3 in chicken and turkey.
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