Prolonged Dietary Selenium Deficiency or Excess Does Not ...

The Journal of Nutrition

Biochemical, Molecular, and Genetic Mechanisms

Prolonged Dietary Selenium Deficiency orExcess Does Not Globally Affect SelenoproteinGene Expression and/or Protein Production inVarious Tissues of Pigs1–3

Yan Liu,4 Hua Zhao,4* Qiaoshan Zhang,4 Jiayong Tang,4 Ke Li,6 Xin-Jie Xia,4,5 Kang-Ning Wang,4 Kui Li,and Xin Gen Lei4,7*

4International Center of Future Agriculture for Human Health, Sichuan Agricultural University, Chengdu, China; 5Institute of

Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China; 6Institute of Animal Science, The Chinese Academy of

Agricultural Science, Beijing, China; and 7Department of Animal Science, Cornell University, Ithaca, NY

Abstract

We previously determined the effects of dietary selenium (Se) deficiency or excess on mRNA abundance of 12

selenoprotein genes in pig tissues. In this study, we determined the effect of dietary Se on mRNA levels of the remaining

porcine selenoprotein genes along with protein production of 4 selenoproteins (Gpx1, Sepp1, Selh, and Sels) and body

glucose homeostasis. Weanling male pigs (n = 24) were fed a Se-deficient (,0.02 mg Se/kg), basal diet supplemented

with 0, 0.3, or 3.0 mg Se/kg as Se-enriched yeast (Angel Yeast) for 16 wk. Although mRNA abundance of the 13

selenoproteins in 10 tissues responded to dietary Se in 3 patterns, there was no common regulation for any given gene

across all tissues or for any given tissue across all genes. Dietary Se affected (P , 0.05) 2, 3, 3, 5, 6, 7, 7, and 8

selenoprotein genes in muscle, hypothalamus, liver, kidney, heart, spleen, thyroid, and pituitary, respectively. Protein

abundance of Gpx1, Sepp1, Selh, and Sels in 6 tissues was regulated (P , 0.05) by dietary Se concentrations in 3 ways.

Compared with those fed 0.3 mg Se/kg, pigs fed 3.0 mg Se/kg became hyperinsulinemic (P , 0.05) and had lower (P ,

0.05) tissue levels of serine/threonine protein kinase. In conclusion, dietary Se exerted no global regulation of gene

transcripts or protein levels of individual selenoproteins across porcine tissues. Pigs may be a good model for studying

mechanisms related to the potential prodiabetic risk of high-Se intake in humans. J. Nutr. 142: 1410–1416, 2012.

Introduction

Selenium (Se) is an essential nutrient for humans and many otherspecies. Classical symptoms of dietary Se deficiency in farm andlaboratory animals were characterized in the 1950s to 1960s (1–8), and supranutrition of Se was found to decrease cancermortality in the 1990s (9–13). However, the underlying molec-ular mechanism for these multiple-phased functions of Se in thebody has remained unclear. Although 24–25 selenoprotein geneswere identified in mammals (14), only a few studies havedetermined collective responses of these genes to dietary Seconcentrations ranging from deficiency to moderately high levels(15–20). Thus, systematic data are needed to link selenoproteingene expression profiles to metabolic impacts of dietary Se

deficiency or excess in tissues. Because pigs are not only animportant food-producing species worldwide but are also anexcellent model for human nutrition (21,22) andmedicine (23,24),we have determined the expression profiles of 12 selenoproteingenes (Gpx1, Gpx2, Gpx4, Dio1, Dio3, Selk, Sep15, Sephs2,Sepn1, Sepp1, Sepw1, and Txnrd1) in liver, testis, thyroid, andpituitary of pigs fed Se-deficient (,0.02 mg/kg), -adequate(0.3 mg/kg), and -excess (3 mg/kg) diets (15). Although dietarySe resulted in dose-dependent increases (P , 0.05) in Seconcentrations and Gpx activities in all of these 4 tissues, itdid not alter mRNA levels of any selenoprotein gene in thyroidor pituitary. More intriguingly, the expression of 7 selenoproteingenes was not affected at all by dietary Se concentrations, even inthe liver. This observed resistance of the assayed selenoproteingenes to dietary Se change has raised the following questions: 1)how dietary Se affects expressions of the remaining selenopro-tein genes in various tissues in addition to the 4 mentionedabove; 2) if feeding longer than 8 wk is needed to show fulleffects of dietary Se on the selenoprotein gene expression intissues, in particular endocrine organs such as thyroid; and 3)what the impact is of dietary Se concentrations on proteinproduction of selected selenoproteins in various tissues.

1 Supported in part by the Chang Jiang Scholars Program of the ChineseMinistry

of Education (X-G.L.), the Chinese Natural Science Foundation (30628019,

30700585, and 30871844), and National Institutes of Health DK 53018 (X-G.L.).2 Author disclosures: Y. Liu, H. Zhao, Q. Zhang, J. Tang, K. Li, X-J. Xia, K-N.

Wang, K. Li, and X. G. Lei, no conflicts of interest.3 Supplemental Tables 1–4 and Figures 1–3 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 jn.nutrition.org

* To whom correspondence should be addressed. E-mail: [email protected] and

[email protected].

ã 2012 American Society for Nutrition.

1410 Manuscript received January 30, 2012. Initial review completed March 10, 2012. Revision accepted May 4, 2012.

First published online June 27, 2012; doi:10.3945/jn.112.159020.Downloaded from https://academic.oup.com/jn/article-abstract/142/8/1410/4630897by gueston 05 February 2018

Recently, a number of human studies have suggested a sur-prising link between high body Se status and adverse blood profilesof glucose and lipid or diabetic susceptibility (25–28). Becausesupranutrition of Se was considered to be anticarcinogenesis (12), itis imperative to clarify if prolonged intake of high dietary Se directlyimpairs glucose homeostasis or insulin function. Although we andothers have attempted to reveal this link in rodents (29–33), pigs arebetter models for humans because of their great digestive andmetabolic similarities to humans (21–24). Two abundant seleno-proteins in tissues and frequently used biomarkers of body Se status(34,35), glutatathione peroxidase-1 (Gpx1) and selenoprotein P(Sepp1) are associated with type-2 diabetes-like phenotypes orinsulin abnormality (36–38). Meanwhile, selenoprotein S (Sels) isalso regulated by glucose (39). Despite no such link reported,selenoproteinH (Selh), a nucleolar thioredoxin-like protein, exhibitsGpx-like activity and distinct expression patterns in highly prolif-erative tissues (40). Because serine/threonine protein kinase (Akt)plays a central role in insulin signaling (41), dysregulation of thisprotein often leads to insulin resistance or malfunction (36).Therefore, we used growing pigs and conducted a 16-wk feedingexperiment to: 1) compare the effects of 0, 0.3, and 3.0 mg Se/kg onmRNA expression profiles of 13 selenoproteins in 10 tissues; 2)determine if the prolonged high Se intake induced dysregulation ofplasma glucose, lipid, and insulin; and 3) explore how tissue proteinlevels ofGpx1, Sepp1, Selh, and Sels, alongwithAkt, were related todietary Se deficiency or excess and the resultant metabolic disorders.

Materials and Methods

Expt. 1Cloning of novel selenoprotein genes in porcine tissues. Because

of the lack of information in the database of National Center forBiotechnology Information (NCBI)8 or other sources, we cloned Selh,Selm, Selv, and Selx to design respective primers for the intended qPCR

analysis. We used a strategy of in silico cloning followed by PCR and

data mining to obtain these genes (42). Primers (Supplemental Table 1)were designed based on homology analysis of the expressed sequence tag

fragment of the target selenoprotein gene with the same gene from other

species or based on the highly conserved region of the gene aligned fromhuman, mouse, rat, and cattle. As previously described (15), the target

genes were amplified by RT-PCR, sequenced 3 times, and aligned using

the BLAST program at NCBI. Total RNA of a Se-adequate, adult male

Landrace pig was isolated from liver to clone Selh, Selm, and Selx andfrom testis to clone Selv.

Expt. 2Animal, diet, and sample collection.Our protocols were approved bySichuan Agriculture University. A total of 24 weanling male pigs (3 wk

old, Duroc-Landrace-Yorkshire crossbred, Swine Research Farm) were

selected and fed a Se-deficient, corn-soybean meal (produced in the Se-

deficient area in Sichuan, China) basal diet (BD) for 3 wk to adjust theirSe status (Table 1). The pigs were divided into 3 groups (n = 8) and fed

the BD supplemented with 0, 0.3, or 3.0 mg Se/kg as Se-enriched yeast

(Angel Yeast) for 16 wk. Two BD were formulated for better meetingnutrient requirements of pigs at body weights #20 kg and .20 kg. The

analyzed Se concentration (mg/kg) was 0.005 for corn, 0.023 for

soybean, 1000 for the Se-rich yeast, 0.02 for pigs at body weights #20

kg, and 0.01 for pigs at body weights.20 kg, respectively. Body weightsof individual pigs were recorded at baseline and then monthly. Blood

samples were collected to prepare plasma samples (15) from individual

pigs (feed deprived overnight for 8 h from 1200 to 0800 of sampling time) at

baseline (wk 0), mid-point (wk 8), and final (wk 16). At the end of the study(wk 16), 6 pigs (feed deprived as described above) from each treatment

group were killed to collect blood, liver, kidney, muscle (longissimus), testis,

thyroid, pituitary, spleen, heart, and hypothalamus (15,16).

Plasma and liver biochemical measures. The hydride generation-

atomic fluorescence spectrometer (AFS-830, Titan Instruments) (43) was

used to measure feed, tissue, and plasma Se concentrations against astandard Se reference (15,16). The activities of Gpx were measured by the

coupled method (44) (0.12 mmol/L of H2O2 as substrate) (15,16). Con-

centrations of protein were determined using the Bradford method (45).

An automated clinical chemistry analyzer (RocheModel 800) was applied,along with respective kit, to determine plasma glucose (Glu-OX kit,

ReeBio), TG (TG-test kit, ReeBio), and insulin (Iodine [125I]Insulin Radio-

immunoassay kit, Groundwork Biotechnology Diagnosticate).

qPCR analyses of the selenoprotein mRNA abundance. Total RNA

was isolated from liver, kidney, muscle, testis, thyroid, pituitary, spleen,

heart, hypothalamus, and blood of 4 pigs from each treatment group(n = 4). The relative mRNA abundances of 16 selenoprotein genes were

assayed. These genes included 12 novel genes of pigs (Diol2, Gpx3,Selh, Seli, Selm, Selo, Sels, Selt, Selv, Selx, Txnrd2, and Txnrd3) and 4

genes (Gpx1, Sep15, Sephs2, and Sepp1) tested in our previous study(15) as inter-experimental controls. However, the results of Txnrd2,Txnrd3, and Diol2 were of poor quality and were not reported herein.

Primers (Supplemental Table 2) for the rest of 13 selenoprotein genesand 2 reference genes, b-actin (Actb) and Gapdh, were designed using

Primer Express 3.0 (Applied Biosystems). The RNA sample prepara-

tion, real time qPCR procedure, and relative mRNA abundance

quantification were the same as previously described by our group(15,16) except for that in the present study, the expression of selenoprotein

gene was set as 1 assuming its application cycle threshold-Ct at 25 and that

of the reference gene Actb at 15.02 (DCt = Cttarget – Ctreference = 25 – 15.02),

and this DCt was then used as the control to normalize the relativeexpression level of target sample genes (2–(DCt sample – DCt control), or 2–DDCt).

To confirm the specific amplification, melting curve and cycle threshold

analyses were performed (Supplemental Figs. 1 and 2).

Western-blot analyses. Tissues were homogenized with the T-per

Tissue Protein Extraction Reagent (Catalog no. 78510, Thermo Fisher

Scientific) and centrifuged at 14,0003 g for 10 min at 48C. The resulting

TABLE 1 Composition of BD as fed1

#20 kg2 .20 kg

g/kg

Corn 564.9 730.5

Soybean 275.0 230.0

Whey powder 120.0 0.0

Choline 1.0 1.0

L-Lysine 3.0 3.2

DL-Methionine 0.2 0.1

L-Threonine 0.6 0.7

Salt 2.0 3.0

CaHPO4 14.0 12.0

CaCO3 7.0 9.0

Trace mineral premix3 10.0 10

Vitamin premix4 0.3 0.5

1 The analyzed Se concentrations of corn, soybean, and Se-rich yeast (Angel Yeast)

were 0.005, 0.023, and 1000 mg/kg, respectively. The analyzed Se concentrations in

the BD were 0.02 and 0.01 mg/kg for #20 and .20 kg, respectively. BD, basal diet.2 Two BD were prepared to better meet the nutrient requirements of pigs at different

ages (weights).3 Trace mineral premix provided/kg diet: FeSO4×7H2O, 993 mg; CuSO4×5H2O, 786 mg;

ZnSO4×7H2O, 440 mg; MnSO4, 27.5 mg; KI, 0.4 mg; and colistin sulfate, 40 mg.4 Vitamin premix provided/kg diet: retinyl acetate, 3027 mg; cholecalciferol, 22 mg; dl-a-

tocopheryl acetate, 32 mg; menadione, 2 mg; thiamin, 4 mg; riboflavin, 14 mg; calcium

pantothenate, 40 mg; niacin, 60 mg; pyridoxol, 6 mg; d-biotin, 0.2 mg; folacin, 1.2 mg;

and cobalamin, 72 mg.

8 Abbreviations used: BD, basal diet; Ct, cycle threshold; NCBI, National Center

for Biotechnology Information.

Dietary selenium and selenoproteins in pigs 1411

Downloaded from https://academic.oup.com/jn/article-abstract/142/8/1410/4630897by gueston 05 February 2018

supernatants of the homogenates (10;40 mg protein/lane) were loaded

onto a SDS-PAGE (12%), transferred to polyvinylidene difluoride

membranes, and incubated with appropriate antibodies (Supplemental

Table 3) as previously described (36).

Statistical analyses. One-way ANOVA followed by Duncan’s multiple

range test (SPSS for Windows 13.0) was used to test effects of the 3dietary Se concentrations on mRNA or protein levels of a given

selenoprotein or Akt within each tissue. Plasma biochemical measures

and growth performance of pigs were analyzed using 1-way ANOVA

with time-repeated measurement. Data are presented as mean 6 SE andthe significance level was set at P , 0.05.

Results

Expt. 1Porcine Selh, Selm, Selv, and Selx were cloned and theirsequences were submitted to NCBI (Table 2). The number ofbasepairs in the gene coding sequence and the number of aminoacids in the deduced peptide sequence were 369 and 123 for Selh,429 and 143 for Selm, 1026 and 342 for Selv, and 348 and 116 forSelx, respectively. There was only one TGA codon for selenocys-teine each in all these 4 genes. The homology of the codingsequences and the deduced peptide sequences in the cloned porcinegenes to their human homologs ranged from 82 to 91% for Selh,Selm, and Selx. However, the cloned porcine Selv exhibited arather low homology of gene coding sequence (70%) and peptidesequence (55%) compared with that of humans.

Expt. 2Growth performance and biochemical measures in plasma

and liver. Pigs fed BD, 0.3 mg Se/kg, and 3.0 mg Se/kg hadsimilar body weights at wk 0 (9.2 6 0.5 kg) and 16 (66.4 6 7.4kg). The 3 groups of pigs also had similar daily feed intakes andgain/feed efficiencies (data not shown). The final plasma and liverSe concentrations were 90 and 86% lower (P , 0.05) in pigs fedBD and 1.4- and 2.7-fold higher (P , 0.05), respectively, in pigsfed 3.0 mg Se/kg than those in pigs fed 0.3 mg Se/kg (Table 3).Compared with the initial values, plasma Gpx activities at wk 8decreased (P, 0.05) by ~50% in pigs fed BD but were more thandoubled (P , 0.05) in those fed 0.3 or 3.0 mg Se/kg. Theseopposite changes led to .5-fold differences in the activities (P ,0.05) between the BD and 2 Se-supplemented groups. At wk 16,pigs fed BD and 3.0 mg Se/kg maintained their respective plasmaGpx activities similar to those at wk 8, but pigs fed 0.3 mg Se/kggroup had a slight decrease. Thus, the final plasma Gpx activitywas 66% lower (P , 0.05) in pigs fed BD and 33% higher (P ,0.05) in pigs fed 3.0 mg Se/kg than that of pigs fed 0.3 mg Se/kg.Likewise, dietary Se deficiency decreased (P , 0.05) liver Gpx

activity by 90% compared with the 0.3 mg Se/kg group. However,increasing dietary Se from 0.3 to 3.0 mg/kg did not produce anyfurther change in the activity. Although final plasma glucoseconcentrations were similar among the 3 groups of pigs, the high-Se diet (3.0 mg/kg) resulted in a 50% higher (P , 0.05) plasmainsulin concentration compared with that of 0.3 mg Se/kg or BD.The final plasma TG concentration in the BD group was 50%lower (P , 0.05) than that of pigs fed 0.3 mg Se/kg.

Abundances of selenoprotein mRNA. Expression of seleno-protein genes in various tissues responded to dietary Se concen-trations in 3 patterns. The first was that dietary Se deficiencyresulted in lower (P , 0.05) tissue mRNA levels compared withthose of supplementing Se at 0.3 and 3.0 mg/kg (Fig. 1). Thesedecreases included Gpx1 in liver, kidney, thyroid, and blood(Fig. 1A); Gpx3 in pituitary and testis (Fig. 1B); Sels in thyroid,pituitary, blood, and spleen (Fig. 1C); and Sepp1 in liver andkidney (Fig. 1D). The second pattern was manifested as higher(P , 0.05) mRNA levels in the BD group than those in the Se-supplemented groups (0.3 and 3.0 mg Se/kg) (Fig. 2). Theseupregulations included Sephs2 (Fig. 2A), Seli (Fig. 2B), and Selx(Fig. 2C) in heart; Selo in hypothalamus and heart (Fig. 2D); Seltin liver, muscle, and heart (Fig. 2E); Sels in kidney and heart (Fig.2F); and Selv in kidney, muscle, and hypothalamus (Fig. 2G).The third way had similar downregulations of mRNA levels byboth BD and 3.0 mg Se/kg compared with 0.3mg Se/kg (Fig. 3).These includedGpx1 in pituitary and spleen (Fig. 3A); Sephs2 inpituitary, thyroid, and hypothalamus (Fig. 3B); Selv in thyroid(Fig. 3C); Selm in thyroid and spleen (Fig. 3D), Seli in thyroid,pituitary, and spleen (Fig. 3E); Selx in spleen (Fig. 3F); Sep15 inthyroid, pituitary, spleen, and kidney (Fig. 3G); and Selh inkidney, thyroid, pituitary, and spleen (Fig. 3H). However,dietary Se deprivation or excess had a nonsignificant effect onthe expression of many selenoprotein genes in various tissues(Supplemental Table 4).

Tissue abundances of selenoproteins. Effects of dietary Seon tissue protein levels of Gpx1 (Fig. 4A), Sepp1 (Fig. 4B), Selh

TABLE 2 Characteristics of the 4 cloned novel pigselenoprotein genes

GeneAccession

no.

Length1Homologous tohuman,2 % Selenocysteine

position3Gene Peptide Gene Peptide

Selh HM018602 369 123 89 89 43

Selm FJ968780 429 143 82 84 45

Selv GQ478346 1026 342 70 55 269

Selx EF113597 348 116 88 91 95

1 The numbers are the base pairs in the coding sequence of the gene and the amino

acids in the deduced peptides.2 As percentage to that of the human gene or peptide compared using BLASTN (57).3 The number indicates its position from the N terminus in the N terminus.

TABLE 3 Effects of dietary Se concentrations on plasmaand liver biochemical indicators of pigs fed theexperimental diets for 16 wk1

Measures BD +0.3 mg Se/kg +3.0 mg Se/kg

Plasma Se, mmol/L

wk 0 0.03 6 0.003 0.03 6 0.01 0.03 6 0.003

wk16 0.02 6 0.01a 0.21 6 0.01b 0.51 6 0.22c

Liver Se, nmol/g tissue

wk 16 0.1 6 0.05a 0.7 6 0.06b 2.6 6 0.91c

Plasma Gpx, U2/mg protein

wk 0 11.6 6 1.2 11.2 6 1.3 9.4 6 1.6

wk 8 5.0 6 2.8a 25.4 6 2.6b 28.5 6 4.0b

wk16 6.3 6 2.3a 19.3 6 2.7b 25.7 6 2.5c

Liver Gpx, U/mg protein

wk 16 28.6 6 7.1a 252 6 67.3b 220 6 31.3b

Plasma glucose, mmol/L

wk 16 5.1 6 0.1 5.6 6 0.7 5.0 6 0.2

Plasma TG, mmol/L

wk 16 0.4 6 0.05a 0.8 6 0.07b 0.6 6 0.09ab

Plasma insulin, pmol/L

wk 16 130 6 3.5a 132 6 13.8a 198 6 31.2b

1 Values are mean6 SE, n = 4–6. Means in a row with superscripts without a common

letter differ, P , 0.05.2 The unit for the enzyme activity is defined as 1 mmol glutathione oxidized per min at 37�C.

1412 Liu et al.


(Fig. 4C), and Sels (Fig. 4D) were also shown in 3 patterns. Thefirst pattern was manifested with lower (P , 0.05) abundancesin the BD group compared with those of pigs fed 0.3 and 3.0 mgSe/kg. These downregulations included Gpx1 in liver andmuscle, Sepp1 in liver, kidney, and muscle, Selh in thyroid andkidney, and Sels in liver, kidney, and muscle. The second pattern

was that tissue selenoproteins further increased (P , 0.05) by3.0 over 0.3 mg Se/kg. These changes included Sepp1 in thyroidand testis, Selh in liver, and Sels in thyroid. The third pattern wasno response of tissue selenoprotein level to dietary Se concen-trations. These included Sepp1 and Sels in heart. Although Gpx1in thyroid, kidney, and testis showed as pattern 1, Sels in testisfell into pattern 3, and Selh in testis increased by 0.3 mg Se/kg,but not 3.0 mg Se/kg, compared with the BD (Supplemental Fig.3A–C), statistical analysis of these changes was impossible dueto the lack of replications. Likewise, the relative distribution ofSelh and Sels in 5–6 tissues was determined in only single assays.Liver Akt protein levels were decreased (P , 0.05) as dietary Seconcentration rose (Fig. 4E). Similar decreases of Akt occurredin thyroid and heart, whereas the opposite change of the proteinoccurred in testis (Supplemental Fig. 3D). However, thesechanges of Akt required further statistical verification.

Discussion

Expression of 13 selenoprotein genes was determined in 10tissues of growing pigs in the present study and illustrated 3types of responses to dietary Se deficiency (BD), adequacy (0.3mg/kg), and excess (3.0 mg/kg). The 3 dietary Se concentrationsexerted the intended impacts on the body Se status of pigs, asshown by plasma and liver Se concentrations and Gpx activities.The successful cloning of Selh, Selm, Selv, and Selx and theavailable information on other novel porcine selenoproteingenes enabled us to design primers for qPCR to complete thesystematic analysis of effects of dietary Se on porcine seleno-protein gene expression profiles initiated in our previous study(15). Specifically, 4 genes (Gpx1, Gpx3, Sels, and Sepp1) weredownregulated in 7 tissues by dietary Se deficiency comparedwith the Se-supplemented groups. These genes seemed to codefor relatively high abundant selenoproteins in the body andtheir responses were consistent with previous reports on pigs(15), chicks (16), mice (17), turkey poults (18), and rats (19).Meanwhile, dietary Se deficiency upregulated mRNA levelsof 7 genes (Seli, Selo, Sels, Selt, Selv, Selx, and Sephs2) in

FIGURE 1 Effects of dietary Se concentrations on mRNA abun-

dance of Gpx1(A), Gpx3 (B), Sels (C), and Sepp1 (D) in various tissues

of pigs at wk 16. Data are mean 6 SE, n = 5. Means for a gene within

a tissue without a common letter differ, P , 0.05.

FIGURE 2 Effects of dietary Se concentrations

on mRNA abundance of Sephs2 (A), Seli (B), Selx

(C), Selo (D), Selt (E), Sels (F), and Selv (G) in various

tissues of pigs at wk 16. Data are mean6 SE, n = 5.

Means for a gene within a tissue without a common

letter differ, P , 0.05. HT, hypothalamus.



5 tissues compared with 0.3 and 3.0 mg Se/kg. Similar up-regulation of Sephs2 expression by Se deficiency was also seenin rat kidney and muscle (19). It remains to be revealed if thistype of upregulation implicates a unique mechanism for vari-ous tissues to prioritize selenoprotein gene expression and/orprotein synthesis under alimited Se supply. Lastly, dietary Sedeficiency and excess, compared with 0.3 mg Se/kg, resulted insymmetric (an inverted U shape) downregulations of 8 genes(Gpx1, Sep15, Sephs2, Selh, Seli, Selm, Selv, and Selx) in 6tissues. In our previous pig study, testis Txnrd1 and Sep15mRNA levels were decreased by 3.0 mg Se/kg compared with

those of 0.3 mg Se/kg. The mirrored impacts by the 2 extremesof dietary Se intake on these 8 genes implied a homeostaticregulation of these genes in response to dietary Se fluctuations.This view and its potential metabolic importance were furthersupported by the fact that tissues involved in this pattern weremainly endocrine organs.

Despite the above-described 3 types of responses, no singleselenoprotein gene exhibited any common response to dietary Seacross various tissues. Meanwhile, no single tissue showed anycommon response to dietary Se across various selenoproteingenes. Therefore, our findings support the view (17) that no

FIGURE 3 Effects of dietary Se

concentrations on mRNA abundance

of Gpx1 (A), Sephs2 (B), Selv (C), Selm

(D), Seli (E), Selx (F), Sep15 (G), and

Selh (H) in various tissues of pigs at

wk 16. Data are mean 6 SE, n = 5.

Means for a gene within a tissue

without a common letter differ, P ,0.05. HT, hypothalamus.

FIGURE 4 Effects of dietary Se concentrations

on protein levels of Gpx1 (A), Sepp1 (B), Selh (C),

Sels (D), and Akt (E) in various tissues of pigs at wk

16. The band was a representative image of 3–5

independent analyses. Values below the protein

band were relative density and are mean 6 SE, n =

3–5. Means without a common letter differ, P ,0.05.

1414 Liu et al.


simple or universal molecular mechanism such as the positionand local context of the UGA codon in the DNA sequence or thesecondary structure of selenocysteine insertion element sequenceand its affinity to binding proteins for a given selenoprotein genecan accurately predict its response to dietary Se regulation.Reciprocally, there is no tissue-specific regulator to controlexpression of different selenoprotein genes in a global way. Inour previous 8-wk feeding experiment (15), mRNA levels of13 genes in thyroid or pituitary were not altered by dietarySe deficiency or excess. In contrast, more selenoprotein genes(7–8 vs. 3–5) were affected by dietary Se in these tissues than inliver or kidney after the 16-wk feeding in the present study.Seemingly, expression of selenoprotein genes in endocrine tissuesmay be resistant to short-term changes of dietary Se intakebut highly responsive to prolonged depletion or overdose ofthe nutrient. This scenario should be considered in searchingfor biomarkers to assess body Se status or in choosing theexperimental duration to determine the effects of dietary Se onendocrine function. While our results illustrated an overrepresen-tation of endocrine tissues in the unique regulatory patterns ofselenoprotein gene expression (an up-regulation or increase by Sedeficiency compared with the Se adequacy; and similar down-regulations or decreases by both dietary Se deficiency and excesscompared with the Se adequacy to form an inverted “U” shapecurve) and protein production (up with high Se, see below), anearlier observation (46) suggested that endocrine tissues were highon the hierarchy of selenoprotein synthesis. The historical aspectsof this hierarchy concept were well elaborated by a recent reviewalong with microarray expression data in rats (20). Mechanis-tically, the high hierarchy of endocrine tissue selenogenometranscripts and proteins, along with their unique responses todietary Se supply, may be associated with their high Se demands(46), special Se transport or uptake (46), and metabolic inter-actions between relevant hormones and Se (24).

The present study revealed novel regulations of protein pro-duction of Gpx1, Sepp1, Selh, and Sels in 6 tissues of pigs by dietarySe. The lack of reagents (antibodies), along with other technicaldifficulties, disallowed much previous research in laboratoryanimals (17,19,32), let alone in large animals like pigs (15), tostudy the regulation of dietary Se beyond gene expression. Thus, theresults from those studies have limited value for functional analysis.In the present study, we identified appropriate antibodies frommultiple sources to assay 4 major selenoproteins that may berelated to body glucose metabolism (see below). Compared withtheir respective mRNA changes, the tissue protein levels of these 4selenoproteins responded to dietary Se concentrations in 3 patterns.The first was that both mRNA and protein showed similar down-regulations by dietary Se deficiency. This type included Gpx1 inliver and muscle, Sepp1 in liver, kidney, and muscle, Selh in thyroidand kidney, and Sels in liver, kidney, and muscle. The second wasthat the protein was elevated by 3.0 over 0.3 mg Se/kg despite aplateau of mRNA at 0.3 mg Se/kg. This type included Sepp1 inthyroid and testis, Selh in liver, and Sels in thyroid. The third wasno change of protein regardless of mRNA responses. This typeincluded Sepp1 in heart and Sels in heart and testis. Altogether, the3 distinct types of responses underscore a disparity or the lack ofsimple regulatory mechanism for selenoproteins by dietary Se in pigtissues.

It was fascinating for us to find that feeding pigs with 3.0 mgSe/kg for 16 wk induced hyperinsulinemia compared with thosefed 0.3 mg Se/kg. More specifically, the Se-overdosed pigs had.50% plasma insulin than the Se-adequate pigs to maintainsimilar plasma glucose concentrations, indicating an early signof insulin resistance. Hyperinsulinemia and insulin resistance

were induced by overexpressing the most abundant selenopro-tein, Gpx1, in mice (36) and feeding gestating rats with highdietary Se (3.0 mg Se/kg) (31). Strikingly, the high-Se inducedhyperinsulinemia was concurrent with a downregulation of Aktprotein in liver and other tissues. Because Akt is a key protein inthe insulin signaling cascade (41), its decrease or dysregulationcould cause insulin resistance (47,48) and thus may serve as oneof the primary mechanisms for the high Se-induced hyperinsuli-nemia (31). Unlike rats (33,49), pigs fed the high-Se diet (3mg/kg)did not develop hyperlipidemia compared with those fed 0.3 mgSe/kg. However, the higher Se diet did result in greater proteinlevels of Gpx1 in heart and testis, Sepp1 in thyroid and testis, Selhin liver and kidney, and Sels in thyroid. Earlier studies indicatethat overexpressing Gpx1 impaired insulin synthesis or sensitivity(36,47), whereas knockout of Gpx1 (48,50) or Sepp1 (37) genesactually improves body insulin sensitivity. In addition, Selh and Selsmight also have the biochemical potential to be involved in glucosemetabolism (39,40,51–53). Although it is unclear if the upregula-tions of tissue Gpx1, Sepp1, Selh, and Sels detected in the presentstudy contributed to the induced hyperinsulinemia and/or thedownregulation of tissue Akt protein, our findings indicate that pigscan be used to model the possible association of high Se intake withincreased risk of diabetes in humans (54). Furthermore, the highsequence homology between pigs and humans in selenoproteingenes reinforces the notion that pigs are a bettermodel than rodentsto study Se metabolism in human health (55,56).

AcknowledgmentsX.G.L. and H.Z. designed the research; Y.L., H.Z., Q.Z., J.T.,Ke Li., X-J.X., K-N.W., Kui Li, and X.G.L. conducted theexperiments and analyzed the data; Y.L, H.Z, and X.G.L. wrotethe paper; and X.G.L. and H.Z. had primary responsibility for thefinal content. All authors read and approved the final manuscript.

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