A Novel Organic Selenium Compound Exerts Unique ...

Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2017/03/29/jn.116.24733 8.DCSupplemental.html

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 Chicks1–3 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 Lei5,8*

Downloaded from jn.nutrition.org at Huazhong Agricultural University on May 6, 2017

4 Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China; 5Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, China; 6Adisseo France S.A.S., Antony, France; 7Department of Veterinary Medicine, Northeast Agricultural University, Harbin, China; and 8Department 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) s and 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, antitumorigenesis, and detoxification (1–6). These metabolic functions of selenium have been attributed mainly to its presence in 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 Demonstration 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.

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: [email protected] (L-H Sun), [email protected] (XG Lei).

ã 2017 American Society for Nutrition. Manuscript received January 3, 2017. Initial review completed January 23, 2017. Revision accepted March 2, 2017. First published online March 29, 2017; doi:10.3945/jn.116.247338.

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Methods Chickens, treatments, and samples collection. Our animal protocol was approved by the Institutional Animal Care and Use Committee of 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-4methylselenobutanoic acid; SepSecS, O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase; SeY, seleno-yeast; tRNA, transfer RNA; TXNRD, thioredoxin reductase.

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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. The experiment lasted for 6 wk. The mortality of birds was 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 measured 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 reductase (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 selenomethionine 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 222ddCt 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 6 SEs. 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


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 muscular 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 regulation of the selenogenome expression in human cancer cell lines by various forms of selenium compounds (30), it was fascinating to determine if SeO exerted unique effects on the expression of the whole selenogenome and selected selenoproteins 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 involves the misacylation of tRNASec by seryl-tRNA synthetase to give Ser-tRNASec. Then, the g-hydroxyl group of Ser-tRNASec is subsequently phosphorylated by O-phosphoseryl-tRNA kinase to give O-phosphoseryl-tRNASec (Sep-tRNASec). Finally, O-phosphoseryltRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes Sep-tRNASec into Sec-tRNASec 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 tRNASec (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 expression 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.

TABLE 1 Effects of 3 selenium forms on growth performances and selenium concentrations in the plasma, liver, and muscle of chicks1 BD Weeks 1–6 Body-weight gain, kg/bird Feed intake, kg/bird Ratio of gain to feed, g/kg Week 3 selenium concentration Plasma,2 μg/L Liver,3 mg/kg Pectoral muscle,3 μg/kg Thigh muscle,2 μg/kg Week 6 selenium concentration Plasma,2 μg/L Liver,3 mg/kg Pectoral muscle,3 μg/kg Thigh muscle,2 μg/kg

2.10 6 0.08 3.45 6 0.10 608 6 12

SeNa 2.15 6 0.02 3.57 6 0.09 603 6 11

SeY 2.16 6 0.04 3.57 6 0.06 606 6 12

SeO 2.21 6 0.07 3.58 6 0.01 617 6 21

110 0.23 67 61

6 6 6 6

6a 0.01a 2a 6a

190 1.7 270 210

6 19b 6 0.07b 6 13b 6 14b

210 1.5 610 280

6 22b 6 0.10b 6 20c 6 17c

230 1.7 740 350

6 21b 6 0.14b 6 34d 6 20d

130 0.32 60 86

6 6 6 6

10a 0.02a 2a 6a

250 2.0 270 210

6 18b 6 0.14b,*,# 6 8b 6 18b

270 2.4 620 300

6 21b 6 0.15b,* 6 22c 6 18c

290 2.5 710 410

6 18b 6 0.21b,# 6 13d 6 40d

#

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 concentration 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 concentrations 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 week 3 and Gpx3 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, and Selenow) and 2 selenocysteine biosynthesisrelated 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) Regulation by an organic selenium compound

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Values are means 6 SEs, 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. 1

TABLE 2 Effect of 3 selenium forms on the redox status in the plasma, liver, and muscles of chicks1 SeNa

SeY

SeO

3.4 6 0.2a 2.8 6 0.4a

4.8 6 0.6b 3.2 6 0.4a

4.8 6 0.4b 1.2 6 0.1b

4.7 6 0.5b 1.0 6 0.2b

47 6 10a

230 6 6.7b

290 6 14c

290 6 22c

7.1 6 0.4a 9.6 6 0.9a

6.9 6 0.3a 6.1 6 0.6b

7.6 6 0.7a 6.1 6 0.4b

9.7 6 0.4b 6.8 6 1.0b

10 6 0.8a 17 6 1.3a

9.8 6 0.4a 14 6 2.1ab

10 6 1.0a 12 6 0.2b

12 6 0.6b 13 6 0.5b

3.5 6 0.4a 0.62 6 0.09a

5.3 6 0.7b 0.58 6 0.05a

5.2 6 0.7b 0.52 6 0.08a,b

5.6 6 0.9b 0.41 6 0.05b

49 6 5.6a 71 6 2.5a

340 6 7.6b 61 6 6.1a,b

410 6 9.7c 68 6 4.2a,b

380 6 8.1c 57 6 3.5b

13 6 0.8a

6.8 6 0.7b

8.6 6 0.4b

8.4 6 0.9b

15 6 0.9a

13 6 0.4ab

12 6 0.5b

13 6 0.9b

Values are means 6 SEs, 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. 1

(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).

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 6 SEs, 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-methylselenobutanoic acid; SeY, BD supplemented with 0.2 mg Se/kg as selenoyeast.

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 792

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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,


Week 3 Plasma GPX, U/mg GSH, μmol/g Liver GPX, U/mg Pectoral muscle TXNRD, U/mg GSH, μmol/g Thigh muscle TXNRD, U/mg GSH, μmol/g Week 6 Plasma GPX, U/mg GSH, U/mg Liver GPX, U/mg GSH, μmol/g Pectoral muscle GSH, μmol/g Thigh muscle GSH, μmol/g

BD

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 selenomethionine 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 expression 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 production, and enzyme activity remains a future research endeavor. From the biochemical standpoint, those uniquely upregulated selenoproteins by SeO are involved in antioxidation, antiinflammation, and detoxification (7, 8, 36–39). Although this

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 6 SEs, 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 supplement with 0.2 mg Se/kg as 2-hydroxy-4-methylselenobutanoic acid; SepSecS, O-phosphoseryltransfer RNA:selenocysteinyltransfer RNA synthase; SeY, base diet supplement with 0.2 mg Se/kg as seleno-yeast; Txnrd, thioredoxin reductase. Regulation by an organic selenium compound

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FIGURE 2 Effect of 3 Se forms on mRNA abundances of selenoprotein 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 6 SEs, 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 supplemented with 0.2 mg Se/kg as 2-hydroxy-4-methylselenobutanoic acid; SepSecS, O-phosphoseryltransfer RNA:selenocysteinyl–transfer RNA synthase; SeY, BD supplemented with 0.2 mg Se/kg as seleno-yeast.

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 needs or growth benefits. 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

FIGURE 5 Effect of 3 Se forms 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 6 SEs, 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.

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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 nonspecifically (41). Technically, selenomethionine represents a selenium 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 increases 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 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 thigh muscle of chicks at weeks 3 (A) and 6 (B). Values are means 6 SEs, 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; 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-phosphoseryltransfer RNA:selenocysteinyl-transfer RNA synthase; SeY, base diet supplement with 0.2 mg Se/kg as selenoyeast; Txnrd, thioredoxin reductase.

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 6 SEs, 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-4methylselenobutanoic acid; SeY, BD supplement with 0.2 mg Se/kg as seleno-yeast.

selenium supplier was the lower relative percentage of selenocysteine 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 accelerated 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 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 6 SEs, 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. Regulation by an organic selenium compound

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concentrations in the muscles and liver were indicative of a poolspecific and time-dependent distribution and saturation of selenium. 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 selenomethionine 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 selenomethionine 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

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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 responsibility for the final content. All authors read and approved the final manuscript.

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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.

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