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The Journal of Nutrition
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
A Novel Organic Selenium Compound Exerts
Unique Regulation of Selenium Speciation,
Selenogenome, and Selenoproteins in
Broiler Chicks
1–3
Ling Zhao,
4,9
Lv-Hui Sun,
4,9
* Jia-Qiang Huang,
5
Mickael Briens,
6
De-Sheng Qi,
4
Shi-Wen Xu,
7
and Xin Gen Lei
5,8
*
4
Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University,
Wuhan, Hubei, China;
5
Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University,
Beijing, China;
6
Adisseo France S.A.S., Antony, France;
7
Department of Veterinary Medicine, Northeast Agricultural University, Harbin,
China; and
8
Department of Animal Science, Cornell University, Ithaca, NY
Abstract
Background: A new organic selenium compound, 2-hydroxy-4-methylselenobutanoic acid (SeO), displayed a greater
bioavailability than sodium selenite (SeNa) or seleno-yeast (SeY) in several species.
Objective: This study sought to determine the regulation of the speciation of selenium, expression of selenogenome and
selenocysteine biosynthesis and degradation-related genes, and production of selenoproteins by the 3 forms of selenium
in the tissues of broiler chicks.
Methods: Day-old male chicks (n= 6 cages/diet, 6 chicks/cage) were fed a selenium-deficient, corn and soy–based diet
[base diet (BD), 0.05 mg Se/kg] or the BD + SeNa, SeY, or SeO at 0.2 mg Se/kg for 6 wk. Plasma, livers, and pectoral and
thigh muscles were collected at weeks 3 and 6 to assay for total selenium, selenomethionine, selenocysteine, redox
status, and selected genes, proteins, and enzymes.
Results: Although both SeY and SeO produced greater concentrations (P< 0.05) of total selenium (20–172%) and of
selenomethionine (#15-fold) in the liver, pectoral muscle, and thigh than those of SeNa, SeO further raised (P< 0.05)
these concentrations by 13–37% and 43–87%, respectively, compared with SeY. Compared with the BD, only SeO
enhanced (P<0.05) the mRNA of selenoprotein (Seleno)sand methionine sulfoxide reductase B1 (Msrb1) in the liver and
thigh (62–98%) and thioredoxin reductase (TXRND) activity in the pectoral and thigh muscles (20–37%) at week 3.
Furthermore, SeO increased (P<0.05) the expression of glutathione peroxidase (Gpx)3, GPX4, SELENOP, and SELENOU
relative to the SeNa group by 26–207%, and the expression of Selenop, O-phosphoseryl-transfer RNA (tRNA):
selenocysteinyl-tRNA synthase, GPX4, and SELENOP relative to the SeY group by 23–55% in various tissues.
Conclusions: Compared with SeNa or SeY, SeO demonstrated a unique ability to enrich selenomethionine and total
selenium depositions, to induce the early expression of Selenos and Mrsb1 mRNA and TXRND activity, and to enhance the
protein production of GPX4, SELENOP, and SELENOU in the tissues of chicks. J Nutr 2017;147:789–97.
Keywords: chick, gene expression, selenium, selenoprotein, speciation
Introduction
Selenium is an essential nutrient for humans and animals, with
potential functions in antioxidant defense, immunity, antitu-
morigenesis, and detoxification (1–6). These metabolic func-
tions of selenium have been attributed mainly to its presence in
selenoproteins as the 21st amino acid, selenocysteine (7). There
are 25–26 selenoprotein genes identified in mammal and avian
species (8–10). The effects of dietary selenium concentrations
2
Author disclosures: L Zhao, L-H Sun, J-Q Huang, D-S Qi, S-W Xu, and XG Lei,
no conflicts of interest. M Briens is an employee of Adisseo.
3
Supplemental Tables 1–7 and Supplemental Figure 1 are available from the
‘‘Online Supporting Material’’ link in the online posting of the article and from the
same link in the online table of contents at http://jn.nutrition.org.
9
These authors contributed equally to this work.
*To whom correspondence should be addressed. E-mail: lvhuisun@mail.hzau.edu.cn
(L-H Sun), xl20@cornell.edu (XG Lei).
1
Supported in part by the Chinese Natural Science Foundation Projects
31501987 and 31320103920; the National Science and Technology Supporting
Program of China Project 2013BAD20B04; the Integration and Demonstrati on for
the Science and Technology Service Mode and Technology of the University
Agriculture in the Modern Great Agricultural Region of Northern Cold Region;
and a research gift by Adisseo France S.A.S.
ã2017 American Society for Nutrition.
Manuscript received January 3, 2017. Initial review completed January 23, 2017. Revision accepted March 2, 2017. 789
First published online March 29, 2017; doi:10.3945/jn.116.247338.
at Huazhong Agricultural University on May 6, 2017jn.nutrition.orgDownloaded from
8.DCSupplemental.html
http://jn.nutrition.org/content/suppl/2017/03/29/jn.116.24733
Supplemental Material can be found at:
on the expression of these genes have been studied in mice (11),
rats (12), pigs (13, 14), chicks (9, 10, 15), and turkeys (16). An
upregulation of 7 selenoprotein genes [glutathione peroxidase
(Gpx)
10
1,Gpx4,selenoprotein (Seleno) k,Selenon,Selenoo,
Selenop, and Selenow] was associated with the protection by
dietary selenium against the occurrence of exudative diathesis in
chicks (9), whereas 6 selenoproteins, namely GPX1, GPX4,
SELENOF, SELENON, SELENOP, and SELENOW, served as
metabolic mediators of body selenium to protect against the
onset of dietary selenium deficiency–induced nutritional mus-
cular dystrophy in chicks (10).
Although forms of both inorganic selenium, such as sodium
selenite (SeNa), and organic selenium, such as seleno-yeast
(SeY), are often used as feed additives in animal diets, the
organic form is the preferred source because of the better
bioavailability (17–22) and lower toxicity (23, 24). A new
organic selenium compound, 2-hydroxy-4-methylselenobutanoic
acid (SeO), has been shown to be more bioavailable than SeNa
or SeY to broilers (25, 26), layers (27, 28), and pigs (29).
Because our previous study demonstrated differential regula-
tion of the selenogenome expression in human cancer cell lines
by various forms of selenium compounds (30), it was fasci-
nating to determine if SeO exerted unique effects on the
expression of the whole selenogenome and selected selenopro-
teins in tissues of chicks compared with the effects of SeNa
and SeY.
Notably, the new form of selenium, SeO, also resulted in greater
enrichment of selenocysteine in the muscles of broilers than did SeY
(22). It is well known that selenocysteine is biosynthesized on
its cognate transfer RNA (tRNA) during selenoprotein synthesis
(31). Briefly, the first step in the selenocysteine formation in-
volves the misacylation of tRNA
Sec
by seryl-tRNA synthetase to
give Ser-tRNA
Sec
. Then, the g-hydroxyl group of Ser-tRNA
Sec
is
subsequently phosphorylated by O-phosphoseryl-tRNA kinase to
give O-phosphoseryl-tRNA
Sec
(Sep-tRNA
Sec
). Finally, O-phosphoseryl-
tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes Sep-tRNA
Sec
into Sec-tRNA
Sec
by using selenophosphate as the selenium donor,
which is the product of selenophosphate synthetases (31, 32).
Meanwhile, selenocysteine insertion sequence–binding protein 2
(SECISBP2) is thought to increase the mRNA of the tRNA
Sec
(32)
and selenocysteine lyase (SCLY) is a selenocysteine degradation
enzyme (33), which play important roles in selenocysteine
metabolism. However, comparative effects of SeO with those
of SeNa and SeY on the expression of these selenocysteine
metabolism-related genes were not studied.
Therefore, this experiment was conducted to determine how
SeO, compared with SeNa and SeY, regulated 1) the deposition of
total selenium, selenomethionine, and selenocysteine; 2) the expres-
sion of the whole selenogenome and 5 key genes related to the
selenocysteine biosynthesis and degradation; and 3) the production
of selected selenoproteins and/or their activity and redox status in
the plasma, liver, and pectoral and thigh muscles of broiler chicks.
Methods
Chickens, treatments, and samples collection. Our animal protocol
was approved by the Institutional Animal Care and Use Committee of
Huazhong Agricultural University, China. In total, 144-d-old male Avian
broilers were randomly allocated to 4 treatment groups with 6 replicates
of 6 birds/cage. The base diet (BD) (Supplemental Table 1) was
composed of corn and soybean produced in the selenium-deficient area
of Sichuan, China, and was not supplemented with selenium. The
other 3 experimental diets were prepared by supplementing the same
BD with 0.2 mg Se/kg as SeNa (Retosel 1% selenium; Retorte GmbH),
SeY (0.2% selenium and 64.9% of selenium as selenomethionine
by analysis; Alkosel and Lallemand), or SeO (Selisseo 2% selenium
and $95% of selenium as SeO by analysis; Adisseo). The analyzed
selenium concentrations in the BD and diets with added SeNa, SeY,
and SeO were 0.048, 0.26, 0.24, and 0.25 mg/kg, respectively. All
birds were allowed free access to the designated diets and distilled
water.Theexperimentlastedfor6wk.Themortalityofbirdswas
monitored daily, whereas body weight and feed intake were measured
weekly. Meanwhile, 6 birds from each treatment group (1 bird/cage)
were killed at weeks 3 and 6 to collect blood, liver, and pectoral and
thigh muscle samples. The samples were washed with ice-cold isotonic
saline before being cut with surgical scissors. The samples were
divided into aliquots, snap-frozen in liquid nitrogen, and stored at
280°C until use (9). Aliquots of liver and pectoral muscle samples
were freeze-dried for analyses of total selenium, selenomethionine,
and selenocysteine.
Antioxidant enzyme activities and selenium, selenomethionine,
and selenocysteine concentrations. As previously described (9),
activities of glutathione peroxidase (GPX) and superoxide dismutase
and concentrations of glutathione and malondialdehyde were mea-
sured by a colorimetric method with the use of specific assay kits
(A005, A001, A006–1, and A003) from the Nanjing Jiancheng
Bioengineering Institute of China. The activity of thioredoxin reduc-
tase (TXNRD) was measured by the NAD(P)H-dependent reduction
of 5,5-dithiobis-(2-nitrobenzoicacid) (6) with the use of a specific
assay kit (BW11) from the Suzhou Comin Biotechnology Co., Ltd. of
China. Protein concentrations were measured by the bicinchoninic
acid assay (14). The concentrations of total selenium in the feed,
plasma, liver, and muscles were measured by the inductively coupled
plasma MS (ICP MS; Agilent 7500cx) (25). Speciation of selenome-
thionine and selenocysteine was carried out as previously described
(25, 34).
Real-time q-PCR and Western blot analyses. Total RNA was
extracted from the liver and muscles (50 mg tissue) of 6 chicks from
each group, and the relative RNA abundance qualification was
conducted as previously described (10, 13). Primers (Supplemental
Table 2) for the assayed genes and the reference gene GAPDH were the
same as those used in our previous study (6). The 22
2ddCt
method was
used for the quantification with GAPDH as a reference gene, and the
relative abundance was normalized to the BD control (as 1). Western
blot analyses of the pertaining samples were performed as previously
described (14). The primary antibodies used for the analyses are
presented in Supplemental Table 3 (10, 35). The specificity and
reliability of individual antibodies against the selected selenoproteins
were validated (Supplemental Figure 1). The abundance of SELENOP
in tissues was estimated based on the intensity of the long band
(57 kDa).
Statistical analysis. Statistical analysis was performed by using SPSS,
version 13. Data are presented as means 6SEs. Dietary effects
were determined by one-factor ANOVA with a significance level of
P< 0.05, and the Tukey-Kramer method was used for multiple mean
comparisons.
Results
Growth performance and deposition of total selenium,
selenomethionine, and selenocysteine. The 4 diets had
similar effects on body-weight gain, feed intake, and the ratio of
gain to feed at week 3 or 6 or overall (Table 1,Supplemental
10
Abbreviations used: BD, base diet; GPX, glutathione peroxidase; Msrb1,
methionine sulfoxide reductase B1; PSTK, O-phosphoseryl-tRNA kinase; SCLY,
selenocysteine lyase; SECISBP2, selenocysteine insertion sequence-binding
protein 2; SELENO, selenoprotein; SeNa sodium selenite; SeO, 2-hydroxy-4-
methylselenobutanoic acid; SepSecS, O-phosphoseryl-tRNA:selenocysteinyl-tRNA
synthase; SeY, seleno-yeast; tRNA, transfer RNA; TXNRD, thioredoxin reductase.
790 Zhao et al.
at Huazhong Agricultural University on May 6, 2017jn.nutrition.orgDownloaded from
Table 4). Compared with the BD, the 3 forms of selenium
enhanced (P< 0.05) selenium concentrations by 73% to 10-fold
in the plasma, liver, and pectoral and thigh muscles at weeks 3
and 6 (Table 1). Compared with SeNa, the 2 organic selenium
compounds SeY and SeO did not further enhance the selenium
concentrations in plasma, but they elevated the selenium concen-
tration by 20–25% (P= 0.06 or 0.08), 1.3- to 1.7-fold (P<0.05)
and 33–95% (P< 0.05) in the liver and pectoral and thigh
muscles, respectively, at week 3 and/or 6. Notably, SeO further
raised the selenium concentrations in the pectoral and thigh
muscles by 15–37% (P< 0.05) compared with SeY.
Compared with the BD, the 2 organic selenium forms SeY
and SeO led to greater (P< 0.05) selenomethionine concentra-
tions in the liver (3.5–7.3-fold) and pectoral muscle (12–19-fold)
at weeks 3 and 6 (Figure 1). Furthermore, SeO resulted in 87%
and 43% greater (P< 0.05) selenomethionine concentrations
in the liver at week 6 and in the pectoral muscle at week 3,
respectively, than did SeY. While all 3 forms of selenium (SeNa,
SeY, and SeO) elevated (P< 0.05) selenocysteine concentrations
in the liver (5.5–9.3-fold) and pectoral muscle (4.5–7.0-fold)
compared with the BD, the elevations by SeNa were 29–46%
and 16–41% greater (P< 0.05) in the liver and pectoral muscle,
respectively, than those by SeY and/or SeO. The concentration
of selenocysteine accounted for >95% in the liver and 84–97%
in the pectoral muscle of the total selenium concentration at
week 3 and/or 6 in the SeNa group but only 64–77% and 30–31%
in the SeY group and 66–74% and 25–29% in the SeO group,
respectively.
Enzyme activity and redox status. Compared with the BD,
the 3 forms of selenium enhanced (P< 0.05) GPX activities in the
plasma and liver by 38–60% and 3.9–7.3-fold, respectively
(Table 2). Notably, the enhancement by the 2 organic selenium
forms SeY and SeO was 11–28% greater (P< 0.05) in the liver
than that by SeNa. Only SeO elevated (P< 0.05) the TXNRD
activities by 37% and 20% in the pectoral and thigh muscles at
week 3, respectively, compared with the BD. Although SeNa
decreased (P< 0.05) glutathione concentration by 36–48% only
in the pectoral muscle compared with the BD, SeO caused
consistent decreases (P< 0.05) in glutathione concentrations in
the plasma, liver, and both muscles. The 4 diets exerted similar
effects on superoxide dismutase activity or malondialdehyde
concentration in the plasma, liver, or muscles (Supplemental
Table 4).
Expression of the selenogenome and selenocysteine
biosynthesis and degradation-related genes. In the liver,
compared with the BD, the 3 forms of selenium enhanced (P<0.05)
mRNA abundance of 11 selenoprotein genes [Gpx1,Gpx3,
Gpx4,methionine sulfoxide reductase B1 (Msrb1),Selenok,
Selenon,Selenop,Selenop2,Selenos,Selenou, and Selenow] and
2 selenocysteine biosynthesis-related genes (Pstk, SepSecS)at
week 3 and/or 6 (Figure 2A, B). Compared with SeNa, SeY
and/or SeO elevated (P< 0.05) mRNA abundance of 6
selenoproteins (Gpx1,Gpx3,Msrb1,Selenop,Selenop2, and
Selenos). Only SeO upregulated (P< 0.05) the hepatic mRNA
abundance of Msrb1 and Selenos compared with the BD at
week3andGpx3 and Selenop2 compared with SeNa and SeY
at week 6.
In the pectoral muscle, compared with the BD, the 3 forms
of selenium enhanced (P< 0.05) mRNA abundance of 8
selenoprotein genes (Gpx1,Gpx3,Gpx4,Selenoh,Selenok,
Selenop,Selenou,andSelenow) and 2 selenocysteine biosynthesis-
related genes (Pstk and SepSecS) at week 3 and/or 6 but
decreased (P< 0.05) Txrnd1 mRNA abundance at week 6
(Figure 3A, B). Compared with SeNa, SeY and/or SeO elevated
(P< 0.05) mRNA abundance of 4 selenoproteins (Gpx3,
Selenop,Selenou, and Selenow) and SepSecS in the pectoral
muscle. Compared with SeY, SeO upregulated (P< 0.05) mRNA
abundance of Selenop at weeks 3 and 6 and SepSecS at week 6 in
the pectoral muscle.
In the thigh muscle, compared with the BD, the 3 forms
of selenium enhanced (P< 0.05) mRNA abundance of 7
selenoprotein genes (Gpx1,Gpx3,Selenoh,Selenom,Selenop,
Selenou,and Selenow) and 2 selenocysteine biosynthesis-related
genes (Pstk and SepSecS) at week 3 and/or 6 but decreased
(P< 0.05) Txrnd1 mRNA abundance at week 6 (Figure 4A, B).
Compared with SeNa, SeY and/or SeO elevated (P< 0.05)
TABLE 1 Effects of 3 selenium forms on growth performances and selenium concentrations in the
plasma, liver, and muscle of chicks
1
BD SeNa SeY SeO
Weeks 1–6
Body-weight gain, kg/bird 2.10 60.08 2.15 60.02 2.16 60.04 2.21 60.07
Feed intake, kg/bird 3.45 60.10 3.57 60.09 3.57 60.06 3.58 60.01
Ratio of gain to feed, g/kg 608 612 603 611 606 612 617 621
Week 3 selenium concentration
Plasma,
2
μg/L 110 66
a
190 619
b
210 622
b
230 621
b
Liver,
3
mg/kg 0.23 60.01
a
1.7 60.07
b
1.5 60.10
b
1.7 60.14
b
Pectoral muscle,
3
μg/kg 67 62
a
270 613
b
610 620
c
740 634
d
Thigh muscle,
2
μg/kg 61 66
a
210 614
b
280 617
c
350 620
d
Week 6 selenium concentration
Plasma,
2
μg/L 130 610
a
250 618
b
270 621
b
290 618
b
Liver,
3
mg/kg 0.32 60.02
a
2.0 60.14
b,
*
,#
2.4 60.15
b,
* 2.5 60.21
b,#
Pectoral muscle,
3
μg/kg 60 62
a
270 68
b
620 622
c
710 613
d
Thigh muscle,
2
μg/kg 86 66
a
210 618
b
300 618
c
410 640
d
1
Values are means 6SEs, n= 6. Labeled means in a row without a common superscript letter differ, P,0.05. *
,#
Different: *P= 0.08,
#
P= 0.06. BD, base diet; SeNa, BD supplemented with 0.2 mg Se/kg as sodium selenite; SeO, BD supplemented with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic acid; SeY, BD supplemented with 0.2 mg Se/kg as seleno-yeast.
2
Selenium concentration was measured in fresh tissues.
3
Selenium concentration was measured in freeze-dried tissues.
Regulation by an organic selenium compound 791
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mRNA abundance of 7 selenoproteins (Gpx3,Msrb1,Selenoh,
Selenom,Selenop,Selenos, and Selenou) and SepSecS in the
thigh muscle at week 3 and/or 6. Compared with SeY, SeO
upregulated (P< 0.05) mRNA abundance of 3 selenoproteins
(Selenoh,Selenop, and Selenos) at week 3 and/or 6 and SepSecS
at week 3 in the thigh muscle. In contrast, mRNA abundance of
the other 3 selenocysteine biosynthesis and degradation-related
genes (Secisbp2,selenophosphate synthetase 1, and Scly) and
the rest of selenoproteins were not affected by the diets or
selenium forms in any of the assayed tissues (Supplemental
Tables 5–7).
Production of selected selenoproteins. Compared with the
BD, the 3 forms of selenium enhanced (P< 0.05) production of
hepatic SELENOP, GPX1, GPX4, SELENOU, and SELENOW
at weeks 3 and 6 (Figure 5A, B). Compared with SeNa, SeY and
SeO enhanced (P< 0.05) production of hepatic SELENOP,
GPX4, and SELENOU at both time points. Compared with SeY,
SeO elevated (P< 0.05) production of hepatic SELENOP and
GPX4 at weeks 3 and 6. Impacts of the 3 forms of selenium on
the production of these 5 selenoproteins in the pectoral (Figure
6A, B) and thigh (Figure 7A, B) muscles at the 2 time points were
very similar to those shown in the liver, with the exception that
SeO resulted in a greater production (P< 0.05) of SELENOU in
the thigh muscle than did SeY at week 3.
Discussion
Our study has demonstrated the unique capacity of SeO, in
comparison with SeNa and SeY, to regulate selenoproteins at the
mRNA, protein, and enzyme activity levels. At both weeks 3 and
6, SeO led to a greater upregulation of Gpx1,Gpx3,Selenop,
Selenoh, and Selenou mRNA; production of GPX4, SELENOP,
FIGURE 1 Effect of 3 Se forms on total Se, SeMet, and SeCys
concentrations in the liver at weeks 3 (A) and 6 (B) and the pectoral
muscle at weeks 3 (C) and 6 (D) in chicks. Values are means 6SEs,
n= 6 for total Se concentrations and n= 3 (pools of 2 chicks) for
SeMet and SeCys concentrations. Means within the same plot
without a common letter differ, P,0.05. A given 2 means within the
same plot labeled with *,
#
,or
+
differ at P= 0.06–0.1. BD, base diet;
DM, dry matter; SeCys, selenocysteine; SeMet, selenomethionine;
SeNa, BD supplemented with 0.2 mg Se/kg as sodium selenite; SeO,
BD supplemented with 0.2 mg Se/kg as 2-hydroxy-4-methylseleno-
butanoic acid; SeY, BD supplemented with 0.2 mg Se/kg as seleno-
yeast.
TABLE 2 Effect of 3 selenium forms on the redox status in the
plasma, liver, and muscles of chicks
1
BD SeNa SeY SeO
Week 3
Plasma
GPX, U/mg 3.4 60.2
a
4.8 60.6
b
4.8 60.4
b
4.7 60.5
b
GSH, μmol/g 2.8 60.4
a
3.2 60.4
a
1.2 60.1
b
1.0 60.2
b
Liver
GPX, U/mg 47 610
a
230 66.7
b
290 614
c
290 622
c
Pectoral muscle
TXNRD, U/mg 7.1 60.4
a
6.9 60.3
a
7.6 60.7
a
9.7 60.4
b
GSH, μmol/g 9.6 60.9
a
6.1 60.6
b
6.1 60.4
b
6.8 61.0
b
Thigh muscle
TXNRD, U/mg 10 60.8
a
9.8 60.4
a
10 61.0
a
12 60.6
b
GSH, μmol/g 17 61.3
a
14 62.1
ab
12 60.2
b
13 60.5
b
Week 6
Plasma
GPX, U/mg 3.5 60.4
a
5.3 60.7
b
5.2 60.7
b
5.6 60.9
b
GSH, U/mg 0.62 60.09
a
0.58 60.05
a
0.52 60.08
a,b
0.41 60.05
b
Liver
GPX, U/mg 49 65.6
a
340 67.6
b
410 69.7
c
380 68.1
c
GSH, μmol/g 71 62.5
a
61 66.1
a,b
68 64.2
a,b
57 63.5
b
Pectoral muscle
GSH, μmol/g 13 60.8
a
6.8 60.7
b
8.6 60.4
b
8.4 60.9
b
Thigh muscle
GSH, μmol/g 15 60.9
a
13 60.4
ab
12 60.5
b
13 60.9
b
1
Values are means 6SEs, n= 6. Labeled means in a row without a common
superscript letter differ, P,0.05. Measures without significant changes were shown
in Supplemental Table 4. BD, BD; GPX, glutathione peroxidase; GSH, glutathione; SeNa,
BD supplemented with 0.2 mg Se/kg as sodium selenite; SeO, BD supplemented with
0.2 mg Se/kg as 2-hydroxy-4-methylselenobutanoic acid; SeY, BD supplemented with
0.2 mg Se/kg as seleno-yeast; TXNRD, thioredoxin reductase.
792 Zhao et al.
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and SELENOU; and GPX activity in the liver, pectoral muscle,
and/or thigh muscle than that by SeNa and/or SeY. At week 3,
only SeO and not SeNa or SeY was able to elevate the expression
of Selenos and Msrb1 mRNA in the liver and thigh muscle and
TXNRD activity in the pectoral and thigh muscles compared
with the BD control. Seemingly, SeO might serve as a novel
selenium supplier or donor that not only shared similar efficacy
with SeNa and SeY in supporting the ‘‘general’’ expression of the
selenogenome but also possessed unique potential in promoting
the functional expression of selected selenoproteins. Previously,
we observed a similar unique upregulation of Gpx1,Gpx4,
Selenof,Selenop,Selenos, and Selenom in human prostate
cancer cells (DU145) by selenium from the selenium-biofortified
porcine serum and methylseleninic acid compared with seleno-
methionine or SeNa (30). Different regulations of selenoprotein
mRNA and protein expression were also produced by SeNa and
SeY in the present study. Although both mRNA and protein
concentrations of GPX1 and SELENOW were upregulated
across the 3 tissues by all the selenium supplements, 11 of the 26
selenoprotein genes were not affected by any form of selenium in
any tissue. This outcome largely resembles the responses of
selenogenome expression to dietary selenium supplementation
in previous studies (9, 10, 15, 16). Indeed, no simple or universal
mechanism has been revealed to explain or predict the global or
specific regulation of selenogenome or selenoprotein expres-
sion in a given tissue by dietary selenium (9, 10, 12–16). Thus,
the mechanism for the unique capacities of SeO in regulating
the identified selenoprotein gene expression, protein produc-
tion, and enzyme activity remains a future research endeavor.
From the biochemical standpoint, those uniquely upregulated
selenoproteins by SeO are involved in antioxidation, anti-
inflammation, and detoxification (7, 8, 36–39). Although this
FIGURE 2 Effect of 3 Se forms
on mRNA abundances of seleno-
protein and SeCys biosynthesis–
related genes relative to the BD
(set at 1.0) in the liver of chicks at
weeks 3 (A) and 6 (B). Values are
means 6SEs, n=6.Means
without a common letter differ,
P,0.05. BD, base diet; Gpx,
glutathione peroxidase; Msrb1,
methionine sulfoxide reductase
B1; Pstk, O-phosphoseryl-transfer
RNA kinase; SeCys, selenocysteine;
Seleno, selenoprotein; SeNa, BD
supplemented with 0.2 mg Se/kg
as sodium selenite; SeO, BD sup-
plemented with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic
acid; SepSecS, O-phosphoseryl-
transfer RNA:selenocysteinyl–transfer
RNA synthase; SeY, BD supple-
mented with 0.2 mg Se/kg as
seleno-yeast.
FIGURE 3 Effect of 3 Se forms
on mRNA abundances of Seleno
and selenocysteine biosynthesis–
related genes relative to the BD
(set at 1.0) in the pectoral muscle
of chicks at weeks 3 (A) and 6 (B).
Values are means 6SEs, n=6.
Labeled means without a common
letter differ, P,0.05. BD, base
diet; Gpx, glutathione peroxidase;
Pstk, O-phosphoseryl-transfer RNA
kinase; Seleno, selenoprotein; SeNa,
BD supplement with 0.2 mg Se/kg
as sodium selenite; SeO, BD sup-
plement with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic
acid; SepSecS, O-phosphoseryl-
transfer RNA:selenocysteinyl-
transf er RNA synthase; SeY, base
diet supplement with 0.2 mg Se/kg
as seleno-yeast; Txnrd, thioredoxin
reductase.
Regulation by an organic selenium compound 793
at Huazhong Agricultural University on May 6, 2017jn.nutrition.orgDownloaded from
type of upregulation resulted in no substantial improvement in
growth performance or redox status of chicks reared at the
conditions of the present study, it may offer extra protection or
benefit to chicks under oxidative, environmental (e.g., heat and
density), and metabolic stresses. Despite no consistent changes
at week 6, the SeO-induced expression of Selenos and Msrb1
mRNA and TXNRD activity in the tissues of chicks at week 3
should not be ignored. Because broiler chicks represent one of
the fastest growing animals during early life, an upregulation of
antioxidant genes or protein can be viewed as the metabolic
needsorgrowthbenefits.
With effects on the chick-growth performance similar to
SeNa or SeY (18, 22, 25, 29), SeO seemed to be more effective in
delivering selenium to enrich tissue selenium after meeting the
need for selenoprotein biosynthesis. First, this hydroxy analogue
of selenomethionine enhanced mRNA, protein, and activity of
selected selenoproteins more than SeNa and SeY did, which is
outlined above. Second, SeO produced the highest selenium
concentrations in both muscles at both time points and the
highest selenomethionine concentrations in the pectoral muscle
at week 3 and in the liver at week 6. These superior efficacies are
consistent with previous reports with broilers, pigs, and cattle
(22, 25, 29, 40). It is well known that selenomethionine
metabolism is closely related to its sulfur homolog and can be
incorporated into proteins in the place of methionine nonspe-
cifically (41). Technically, selenomethionine represents a sele-
nium storage form that could compete with methionine for
absorption and protein synthesis. However, the total methionine
concentration was 0.69% in the BD, whereas the dietary
incorporation of SeY and SeO was at 0.01% and 0.001%,
respectively, to supply the required selenium (0.2 mg/kg). The
extremely low molar ratios of selenomethionine to methionine
(1:21,200 and 1:14,500 for SeY and SeO, respectively) in the
diets probably precluded a major effect of selenomethionine on
methionine metabolism. Because SeO caused no further in-
creases in the plasma total selenium concentrations compared
with those caused by SeNa and SeY at either time point, the
resultant differences in total selenium and selenomethionine
FIGURE 4 Effectof3Seforms
on mRNA abundances of Seleno
and selenocysteine biosynthesis–
related genes relative to the BD
(set at 1.0) in the thigh muscle of
chicks at weeks 3 (A) and 6 (B).
Values are means 6SEs, n=6.
Means without a common letter
differ, P,0.05. BD, base diet; Gpx,
glutathione peroxidase; Msrb1,me-
thionine sulfoxide reductase B1;
Pstk, O-phosphoseryl-transfer RNA
kinase; Seleno, selenoprotein;
SeNa, BD supplement with 0.2 mg
Se/kg as sodium selenite; SeO, BD
supplement with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic
acid; SepSecS, O-phosphoseryl-
transfer RNA:selenocysteinyl-transfer
RNA synthase; SeY, base diet sup-
plement with 0.2 mg Se/kg as seleno-
yeast; Txnrd, thioredoxin reductase.
FIGURE 5 Effectof3Seforms
on protein production of SELENOP,
GPX1, GPX4, SELENOW, and
SELENOU relative to the BD (set
at 100) in the liver of chicks at
weeks 3 (A) and 6 (B). Values are
means 6SEs, n= 3–4. The relative
density values under respective
bands without a common letter
differ, P,0.05. ACTB, b-actin;
BD, base diet; GPX, glutathione
peroxidase; SELENO, selenoprotein;
SeNa, BD supplement with 0.2 mg
Se/kg as sodium selenite; SeO, BD
supplement with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic
acid; SeY, BD supplement with
0.2 mg Se/kg as seleno-yeast.
794 Zhao et al.
at Huazhong Agricultural University on May 6, 2017jn.nutrition.orgDownloaded from
concentrations in the muscles and liver were indicative of a pool-
specific and time-dependent distribution and saturation of se-
lenium. From the metabolic modeling standpoint (42, 43),
plasma selenium represents the mobile pool of body selenium
that circulates selenium to meet various metabolic needs in
tissues and is often maintained at a steady state with an adequate
selenium supply. Clearly, the 3 forms of selenium shared similar
efficacy in maintaining the plasma selenium pool. The liver has
the tissue that synthesizes SELENOP that is supposed to carry
selenium to other tissues, which serves as the major selenium
metabolism pool (43–45). The dynamic nature of this selenium
pool and the metabolic priority of selenium partitioning may
help to explain why the difference in total selenium and se-
lenomethionine concentrations between the SeY and SeO groups
appeared only at the later time point. Obviously, muscle
functions as the largest deposit pool of selenium (44, 45) and
showed the highest enrichment of selenium and/or selenomethi-
onine at the earlier time point.
The superior efficacy of the organic selenium (SeY and SeO)
to the inorganic selenium (SeNa) in enriching total selenium and
selenomethionine in the liver and muscles (21, 22) may be
associated with the mode of intestinal absorption (46, 47) and
the ability to be incorporated into proteins in the place of
methionine (22, 41). Additional evidence that SeO is a better
selenium supplier was the lower relative percentage of seleno-
cysteine to the higher total selenium in the liver and pectoral
muscle compared with that of SeNa. Although SeNa produced
slightly higher concentrations of selenocysteine in both tissues
than SeO did, the relative percentages of selenocysteine to the
total selenium were >95% in the liver and 84–97% in the
muscles for SeNa, but only 66–74% and 25–29% for SeO,
respectively. If selenocysteine is considered to be more a
functional form and selenomethionine to be more a storage
form, the higher percentages of selenocysteine to the lower
total selenium concentration in the SeNa group than the in SeO
group may be interpreted as less saturation of the functional
selenium for the biosynthesis of selenoproteins. In fact, SeO
resulted in greater protein productions of GPX4, SELENOP,
and/or SELENOU and TXNRD activity than did SeNa or SeY.
The moderately elevated selenocysteine concentrations by
SeNa compared with SeO may be paradoxically unutilized as
free selenocysteine or ‘‘selenocysteine-containing proteins,’’
which are not incorporated into selenoproteins or an acceler-
ated selenoprotein degradation (48–50). New antibodies will
be required to determine if the elevated selenocysteine by SeNa
promotes the production of other selenoproteins not assayed in
the presented study. However, our results were inconsistent
with previous studies in which higher muscle selenocysteine
FIGURE 6 Effect of 3 Se forms
on protein production of SELENOP,
GPX1, GPX4, SELENOW, and
SELENOU relative to the BD (set
at 100) in the pectoral muscle of
chicks at weeks 3 (A) and 6 (B).
Values are means 6SEs, n= 3–4.
The relative density values under
respective bands without a com-
mon letter differ, P,0.05. ACTB,
b-actin; BD, base diet; GPX, glu-
tathione peroxidase; SELENO,
selenoprotein; SeNa, BD supple-
ment with 0.2 mg Se/kg as sodium
selenite; SeO, BD supplement
with 0.2 mg Se/kg as 2-hydroxy-4-
methylselenobutanoic acid; SeY,
BD supplement with 0.2 mg Se/kg
as seleno-yeast.
FIGURE 7 Effect of 3 Se forms
on protein production of SELENOP,
GPX1, GPX4, SELENOW, and
SELENOU relative to the BD (set
at 100) in the thigh muscle of
chicks at weeks 3 (A) and 6 (B).
Values are means 6SEs, n= 3–4.
The relative density values under
respective bands without a com-
mon letter differ, P,0.05. ACTB,
b-actin; BD, base diet; GPX, gluta-
thione peroxidase; SELENO, seleno-
protein; SeNa, BD supplement with
0.2 mg Se/kg as sodium selenite; SeO,
BD supplement with 0.2 mg Se/kg as
2-hydroxy-4-methylselenobutanoic
acid; SeY, BD supplement with 0.2 mg
Se/kg as seleno-yeast.
Regulation by an organic selenium compound 795
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concentrations were produced by SeY than by SeNa in lambs,
as well as by SeO than by SeY in broilers (21, 22). These divergences
remain to be explained.
It is novel, to the best of our knowledge, to reveal the elevated
mRNA expression of 2 selenocysteine biosynthesis–related
genes, Pstk and SepSecS, by all 3 forms of selenium in the 3
tissues. This upregulation was largely consistent with their
positive effects on the selenocysteine concentrations and the
functional expression of selenoproteins at the mRNA, protein,
and activity levels. However, there were 2 subtle discrepancies
associated with this finding. Although SeO or SeY led to slightly
lower concentrations of selenocysteine in the liver and/or pectoral
muscle than SeNa, the 2 organic forms of selenium actually induced
similar or greater expression of Pstk and SepSecS. This may imply
a complex feedback mechanism in regulating selenocysteine
biosynthesis (51). It is intriguing that the other 3 selenocysteine
biosynthesis–related genes, selenophosphate synthetase 1,Sephs2,
and Secisbp2, and the selenocysteine-degrading enzyme gene Scly
failed to respond to the selenium supplementation of any form. It
warrants future research to find out if these proteins are regulated
by dietary selenium at the posttranscriptional sites.
In contrast to those upregulated selenoprotein genes, the
Txrnd1 mRNA abundances in the muscles were decreased by the
3 forms of selenium compared with the BD at week 6. This type
of downregulation was shown in previous studies (9, 13).
Furthermore, concentrations of glutathione in plasma, the liver,
and/or muscle were actually inversely related to the elevated
TXRND activity in the muscle by SeO at week 3 and GPX
activity in the liver by SeO and SeY at weeks 3 and 6. Because
selenium deficiency stimulated hepatic glutathione synthesis and
release to blood (52), the decreased glutathione in the SeO or
SeY group may be interpreted as an adaptation or coordination
to the elevated production of GPX and other antioxidant
selenoproteins.
Acknowledgments
We thank Rong-Wei Tang, Xuan Fang, Zeng-Quan Wei,
Zhi-Yuan Zhao, Guan-Jun Ma, and Shahid Ali Rajput for
technical assistance. L-HS, J-QH, and XGL designed the
research; LZ, L-HS, MB, S-WX, and D-SQ conducted the
experiments and analyzed the data; LZ, L-HS, J-QH, and XGL
wrote the manuscript; and L-HS and XGL had primary respon-
sibility for the final content. All authors read and approved the
final manuscript.
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