Dietary Selenium Across Species.

This review traces the discoveries that led to the recognition of selenium (Se) as an essential nutrient and discusses Se-responsive diseases in animals and humans in the context of current understanding of the molecular mechanisms of their pathogeneses. The article includes a comprehensive analysis of dietary sources, nutritional utilization, metabolic functions, and dietary requirements of Se across various species. We also compare the function and regulation of selenogenomes and selenoproteomes among rodents, food animals, and humans. The review addresses the metabolic impacts of high dietary Se intakes in different species and recent revelations of Se metabolites, means of increasing Se status, and the recycling of Se in food systems and ecosystems. Finally, research needs are identified for supporting basic science and practical applications of dietary Se in food, nutrition, and health across species.

Gene Set Enrichment Analysis of Selenium-Deficient and High-Selenium Rat Liver Transcript Expression and Comparison With Turkey Liver Expression.

BACKGROUND: Better biomarkers of selenium (Se) status and a better understanding of toxic Se biochemistry are needed to set safe dietary upper limits. In previous studies, differential expression (DE) of individual liver transcripts in rats and turkeys failed to identify a single transcript that was consistently and significantly (q < 0.05) altered by high Se. OBJECTIVES: To evaluate the effect of Se status on rat liver transcript expression data at the level of gene sets, and to compare transcript expression in rats with that in turkeys to identify common regulated transcripts. METHODS: Gene set enrichment analysis (GSEA) was conducted on liver from weanling rats fed an Se-deficient basal diet (0.005 µg Se/g) supplemented with 0, 0.24 (Se-adequate), 2, or 5 µg Se/g diet as selenite for 28 d. In addition, transcript expression was compared with liver expression in turkeys fed 0, 0.4, 2, or 5 µg Se/g diet as selenite. RESULTS: Se deficiency significantly downregulated the rat selenoprotein gene set but also upregulated gene sets for a variety of pathways, processes, and disease states. GSEA of 2 compared with 0.24 µg Se/g found no significantly up- or downregulated gene sets, showing that 2 µg Se/g is not particularly toxic to the rat. GSEA analysis of 5 compared with 0.24 µg Se/g transcripts, however, found 27 significantly upregulated gene sets for a wide variety of conditions. Cross-species GSEA comparison of transcript expression, however, identified no common gene sets significantly and consistently regulated by high Se in rats and turkeys. In addition, comparison of individual marginally significant (unadjusted P < 0.05) DE transcripts between rats and turkeys also failed to find common transcripts. CONCLUSIONS: The dramatic increase in significant liver transcript DE and GSEA gene sets in rats fed 5 compared with 2 µg Se/g clearly appears to be a biomarker for Se toxicity, albeit not Se-specific. These analyses, however, failed to identify specific transcripts or pathways, biological states, or processes that were directly linked with high Se status, strongly indicating that adaptation to high Se lies outside transcriptional regulation.

Selenoprotein Gene Nomenclature.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Effect of graded levels of selenium supplementation as selenite on expression of selenosugars, selenocysteine, and other selenometabolites in rat liver.

Using high pressure liquid chromatography (HPLC) coupled with selenium-specific inductively coupled plasma mass spectrometry (ICP-MS) and molecule specific (Orbitrap MS/MS) detection, we previously found that far more selenium (Se) is present as selenosugar (seleno-N-acetyl galactosamine) in Se-adequate turkey liver than is present as selenocysteine (Sec) in true selenoproteins, and that selenosugars account for half of the Se in high-Se turkey liver. To expand these observations to mammals, we studied Se metabolism in rats fed graded levels of selenite from 0 to 5 µg Se/g for 4 wk. In Se-adequate (0.24 µg Se/g) rats, 43% of liver Se was present as Sec, 32% was present as selenosugars, and 22% as inorganic Se bound to protein. In liver of rats fed 5 µg Se/g as selenite, the quantity of Sec remained at the Se-adequate plateau (11% of total Se), 22% was present as low molecular weight (LMW) selenosugars with substantial additional selenosugars linked to protein, but 64% was present as inorganic Se bound to protein. No selenomethionine was found at any level of selenite supplementation. Below the Se requirement, Se is preferentially incorporated into Sec-selenoproteins. Above the dietary Se requirement, selenosugars become by far the major LMW water soluble Se species in liver, and levels of selenosugar-decorated proteins are far higher than Sec-selenoproteins, making these selenosugar-decorated proteins the major Se-containing protein species in liver with high Se supplementation. This accumulation of selenosugars linked to cysteines on proteins or the build-up of inorganic Se bound to protein may underlie Se toxicity at the molecular level.

Selenomethionine supplementation and expression of selenosugars, selenocysteine, and other selenometabolites in rat liver.

Selenomethionine (SeMet) as a methionine analog can be incorporated into protein. In turkeys, we recently found that selenium (Se) as selenite is not metabolized to SeMet but rather to selenosugars (seleno-N-acetyl galactosamine) bound to protein as well as to selenocysteine (Sec) in selenoproteins. To characterize the metabolism of SeMet, we fed rats graded levels of SeMet from 0 to 5 µg Se/g in a Se-deficient diet for 4 wk, and investigated the fate and accumulation of liver Se using high pressure liquid chromatography (HPLC) coupled with Se-specific inductively coupled plasma mass spectrometry (ICP-MS) and molecule specific (Orbitrap MS/MS) detection. Up to 0.24 µg Se/g (Se requirement for maximal glutathione peroxidase activity), Sec accounted for ∼40% of total liver Se whereas SeMet only accounted for 3-11%. Analysis of water-soluble extracts found negligible low molecular weight (LMW) Se species in rats fed 0 and 0.08 µg Se/g, including no SeMet. At 0.24 µg Se/g and above, SeMet accounted for only 10% of LMW Se species, whereas methyl- and glutathionyl-selenosugars accounted for 70% of LMW Se species. Above the Se requirement, SeMet was ∼30% of the proteinaceous amino acids, whereas Sec levels fell to 5% in rats fed 5 µg Se/g as SeMet. Last, considerably less inorganic Se was bound to liver protein with high SeMet as compared to selenite in a parallel study. SeMet is efficiently metabolized and mixes with the common Se metabolite pool, where Se is preferentially incorporated into Sec and Sec-selenoproteins until selenoproteins plateau; with high SeMet intake, Se is increasingly accumulated as LMW selenosugars and as selenosugar-decorated proteins.

Valuing the Diversity of Research Methods to Advance Nutrition Science.

The ASN Board of Directors appointed the Nutrition Research Task Force to develop a report on scientific methods used in nutrition science to advance discovery, interpretation, and application of knowledge in the field. The genesis of this report was growing concern about the tone of discourse among nutrition professionals and the implications of acrimony on the productive study and translation of nutrition science. Too often, honest differences of opinion are cast as conflicts instead of areas of needed collaboration. Recognition of the value (and limitations) of contributions from well-executed nutrition science derived from the various approaches used in the discipline, as well as appreciation of how their layering will yield the strongest evidence base, will provide a basis for greater productivity and impact. Greater collaborative efforts within the field of nutrition science will require an understanding that each method or approach has a place and function that should be valued and used together to create the nutrition evidence base. Precision nutrition was identified as an important emerging nutrition topic by the preponderance of task force members, and this theme was adopted for the report because it lent itself to integration of many approaches in nutrition science. Although the primary audience for this report is nutrition researchers and other nutrition professionals, a secondary aim is to develop a document useful for the various audiences that translate nutrition research, including journalists, clinicians, and policymakers. The intent is to promote accurate, transparent, verifiable evidence-based communication about nutrition science. This will facilitate reasoned interpretation and application of emerging findings and, thereby, improve understanding and trust in nutrition science and appropriate characterization, development, and adoption of recommendations.

Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents.

BACKGROUND: Protein and mRNA levels for several selenoproteins, such as glutathione peroxidase-1 (Gpx1), are down-regulated dramatically by selenium (Se) deficiency. These levels in rats increase sigmoidally with increasing dietary Se and reach defined plateaus at the Se requirement, making them sensitive biomarkers for Se deficiency. These levels, however, do not further increase with super-nutritional or toxic Se status, making them ineffective for detection of high Se status. Biomarkers for high Se status are needed as super-nutritional Se intakes are associated with beneficial as well as adverse health outcomes. To characterize Se regulation of the transcriptome, we conducted 3 microarray experiments in weanling mice and rats fed Se-deficient diets supplemented with up to 5 µg Se/g diet. RESULTS: There was no effect of Se status on growth of mice fed 0 to 0.2 µg Se/g diet or rats fed 0 to 2 µg Se/g diet, but rats fed 5 µg Se/g diet showed a 23% decrease in growth and elevated plasma alanine aminotransferase activity, indicating Se toxicity. Rats fed 5 µg Se/g diet had significantly altered expression of 1193 liver transcripts, whereas mice or rats fed ≤ 2 µg Se/g diet had < 10 transcripts significantly altered relative to Se-adequate animals within an experiment. Functional analysis of genes altered by Se toxicity showed enrichment in cell movement/morphogenesis, extracellular matrix, and development/angiogenesis processes. Genes up-regulated by Se deficiency were targets of the stress response transcription factor, Nrf2. Multiple regression analysis of transcripts significantly altered by 2 µg Se/g and Se-deficient diets identified an 11-transcript biomarker panel that accounted for 99% of the variation in liver Se concentration over the full range from 0 to 5 µg Se/g diet. CONCLUSION: This study shows that Se toxicity (5 µg Se/g diet) in rats vastly alters the liver transcriptome whereas Se-deficiency or high but non-toxic Se intake elicits relatively few changes. This is the first evidence that a vastly expanded number of transcriptional changes itself can be a biomarker of Se toxicity, and that identified transcripts can be used to develop molecular biomarker panels that accurately predict super-nutritional and toxic Se status.

Metabolism of Tracer 75Se Selenium From Inorganic and Organic Selenocompounds Into Selenoproteins in Rats, and the Missing 75Se Metabolites.

We now know much about selenium (Se) incorporation into selenoproteins, and there is considerable interest in the optimum form of Se for supplementation and prevention of cancer. To study the flux of 75Se into selenoprotein, rats were fed 0 to 5 µg Se/g diet as selenite for 50-80 d and injected iv with 50 µCi of 75Se-labeled selenite, selenate, selenodiglutathione, selenomethionine, or selenobetaine at tracer levels (~0.5 µg Se). The rats were killed at various times and 75Se incorporation into selenoproteins was assessed by SDS/PAGE. These studies found that there is very rapid Se metabolism from this diverse set of selenocompounds to the common intermediate used for synthesis and incorporation of 75Se into the major selenoproteins in a variety of tissues. No selenocompound was uniquely or preferentially metabolized to provide Se for selenoprotein incorporation. Examination of the SDS/PAGE selenoprotein profiles, however, reveals that synthesis of selenoproteins is only part of the full Se metabolism story. The 75Se missing from the selenoprotein profiles, especially at early timepoints, is likely to be both low-MW and high-MW selenosugars and related precursors, as we recently found in livers of turkeys fed Se-adequate and high-Se diets. Differential metabolism of different selenocompounds into different selenosugar species may occur; these species may be involved in prevention of cancer or other diseases linked to Se status and may be associated with Se toxicity. Additional studies using HPLC-mass spectroscopy will likely be needed to fully flesh out the complete metabolism of selenium.

Differential protein expression due to Se deficiency and Se toxicity in rat liver.

There is a U-shaped dose-response between selenium (Se) status and health outcomes, but underlying metabolic processes are unclear. This study aims to identify candidate proteins in liver regulated by dietary Se, ranging from deficiency to toxic. Male rats (n=4) were fed graded Se concentrations as selenite for 28 days. Bulk Se analysis was performed by ICP-MS on both soluble and insoluble fractions. Soluble fraction samples were chromatographically separated for identification of selenocompounds by SEC-ICP-MS and protein quantification by LC-MS/MS. Bioinformatics analysis compared low-Se (0 and 0.08 µg Se g-1) and high-Se (0.8, 2 and 5 µg Se g-1) with adequate-Se (0.24 µg Se g-1) diets. Major breakpoints for Se were seen at 0.8 and 2 µg Se g-1 in the insoluble and soluble fractions, respectively. Glutathione peroxidase 1 protein abundance reached a plateau at ≥0.08 µg Se g-1diet; Se bound to selenium binding protein 2 was observed with 2 and 5 µg Se g-1 Se. The extreme diets presented the highest number of differentially expressed (P value <0.05, FC ≥1.2) proteins in comparison to the adequate-Se diet (0 µg Se g-1: 45 proteins; 5 µg Se g-1: 59 proteins); 13 proteins were commonly affected in 0 and 5 µg Se g-1 treatments. Network analysis revealed that the metabolism of glutathione, xenobiotics and amino acids were enriched in both 0 and 5 µg Se g-1 diets, indicating a U-shape effect of Se. This similarity is likely due to down-stream effects of lack of essential selenoproteins in Se deficiency and due to toxic effects of Se that exceeds the capacity to cope with excess Se.

Correction: Identification and determination of selenocysteine, selenosugar, and other selenometabolites in turkey liver.

Correction for 'Identification and determination of selenocysteine, selenosugar, and other selenometabolites in turkey liver' by Katarzyna Bierla et al., Metallomics, 2020, DOI: 10.1039/d0mt00040j.

Identification and determination of selenocysteine, selenosugar, and other selenometabolites in turkey liver.

Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. To further study selenometabolites in Se-deficient, Se-adequate, and high-Se liver, turkey poults were fed 0, 0.4, and 5 µg Se g-1 diet as Na2SeO3 (Se(iv)) in a Se-deficient (0.005 µg Se g-1) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. No selenomethionine (SeMet) was detected in liver in turkeys fed either this true Se-deficient diet or supplemented with inorganic Se, showing that turkeys cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient liver, as expected in animals with negligible selenoprotein levels. Sec content in high Se liver only doubled as compared to Se-adequate liver, indicating that the 6-fold increase in liver Se was not due to increases in selenoproteins. What increased dramatically in high Se liver were low molecular weight (MW) selenometabolites: glutathione-, cysteine- and methyl-conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). Substantial Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide bonds. In high-Se liver, these "selenosugar-decorated" proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to low MW selenometabolites. In summary, more Se is present as the selenosugar moiety in Se-adequate liver, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols.

The hepatic transcriptome of the turkey poult (Meleagris gallopavo) is minimally altered by high inorganic dietary selenium.

There is interest in supplementing animals and humans with selenium (Se) above Se-adequate levels, but the only good biomarker for toxicity is tissue Se. We targeted liver because turkeys fed 5 µg Se/g have hepatic Se concentrations 6-fold above Se-adequate (0.4 µg Se/g) levels without effects on growth or health. Our objectives were (i) to identify transcript biomarkers for high Se status, which in turn would (ii) suggest proteins and pathways used by animals to adapt to high Se. Turkey poults were fed 0, 0.025, 0.4, 0.75 and 1.0 µg Se/g diet in experiment 1, and fed 0.4, 2.0 and 5.0 µg Se/g in experiment 2, as selenite, and the full liver transcriptome determined by RNA-Seq. The major effect of Se-deficiency was to down-regulate expression of a subset of selenoprotein transcripts, with little significant effect on general transcript expression. In response to high Se intake (2 and 5 µg Se/g) relative to Se-adequate turkeys, there were only a limited number of significant differentially expressed transcripts, all with only relatively small fold-changes. No transcript showed a consistent pattern of altered expression in response to high Se intakes across the 1, 2 and 5 µg Se/g treatments, and there were no associated metabolic pathways and biological functions that were significant and consistently found with high Se supplementation. Gene set enrichment analysis also found no gene sets that were consistently altered by high-Se and supernutritional-Se. A comparison of differentially expressed transcript sets with high Se transcript sets identified in mice provided high Se (~3 µg Se/g) also failed to identify common differentially expressed transcript sets between these two species. Collectively, this study indicates that turkeys do not alter gene expression in the liver as a homeostatic mechanism to adapt to high Se.

Transcript analysis of the selenoproteome indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity.

Dietary selenium (Se) requirements in rats have been based largely upon glutathione peroxidase-1 (Gpx1) enzyme activity and Gpx1 mRNA levels can also be used to determine Se requirements. The identification of the complete selenoprotein proteome suggests that we might identify additional useful molecular biomarkers for assessment of Se status. To characterize Se regulation of the entire rat selenoproteome, weanling male rats were fed a Se-deficient diet (<0.01 microg Se/g) supplemented with graded levels of Se (0-0.8 microg/g diet) for 28 d, Se status was determined by tissue Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver, kidney, and muscle was determined by quantitative real-time-PCR. Tissue Se and selenoenzyme biomarkers indicated that minimal Se requirements were

Selenium requirements are higher for glutathione peroxidase-1 mRNA than gpx1 activity in rat testis.

Selenium (Se) plays a critical role in testis, sperm, and reproduction, and testis Se levels are remarkably maintained in Se deficiency. In most other tissues, Se levels decrease dramatically as do levels of most selenoproteins and levels of a subset of Se-regulated selenoprotein mRNAs. Because of the recent identification of key molecules in the targeted trafficking of Se to the testis, we examined the hierarchy of Se regulation in testis by determining the dietary Se regulation of the full testis selenoproteome in rats fed graded levels of Se (0 to 0.8 microg Se/g) as Na2SeO3 for 28 d. Se status did not significantly affect testis weight or glutathione peroxidase 4 (Gpx4) activity (P>0.05). qRT-PCR analysis of selenoprotein mRNA expression revealed that 21 of the 24 selenoprotein mRNAs and ApoER2 mRNA (the selenoprotein P [Sepp1] receptor) were also not regulated significantly by dietary Se status. In contrast, Gpx1 activity decreased to 28% of Se-adequate levels, and mRNA levels for Gpx1, Sepp1, and Sepw1 (selenoprotein W) decreased significantly in Se-deficient rats to 45, 46, and 55%, respectively, of Se-adequate plateau levels. Overlap of hyperbolic Gpx4 activity and Sepw1 mRNA response curves with testis Se concentration, all with minimum dietary Se requirements<0.016 microg Se/g, showed the priority for synthesis of Gpx4. Higher minimum dietary Se requirements of 0.04 microg Se/g for Gpx1 activity and Sepp1 mRNA, and the even higher minimum dietary Se requirement of 0.08 microg Se/g for Gpx1 mRNA, suggest that the hierarchy of these biomarkers reflects distinct, lower priority pools, cell types, and roles for Se within the testis.

High dietary inorganic selenium has minimal effects on turkeys and selenium status biomarkers.

The current NRC turkey Se requirement is 0.2 µg Se/g diet. Recent studies evaluating tissue Se, glutathione peroxidase (GPX) activities, and selenoprotein transcript expression concluded that the dietary Se requirement of the turkey poult should be raised to 0.4 µg Se/g when supplemented with inorganic Se. The FDA currently limits Se inclusion in premixed diets for poultry and other livestock species to 0.3 µg Se/g diet. Thus, there is a need to investigate the effect of high dietary Se (>1.0 µg Se/g) in turkeys. The present study fed turkey poults 2 and 5 µg Se/g diet to characterize tissue Se accumulation in turkey poults fed high dietary inorganic Se and to evaluate the efficacy of selenoprotein activity and transcript expression as biomarkers of high Se status. Day-old male poults were fed 0.4, 2, or 5 µg Se/g for 28 d. There was no significant effect of Se supplementation on poult growth. Supplementation with 5 µg Se/g diet resulted in Se concentrations that were 5.6X, 1.7X, 1.9X, and 2.0X greater than Se-adequate levels in liver, kidney, breast, and thigh, respectively, and GPX activities in plasma, red cells, liver, kidney, and heart that were ≤2.0X Se-adequate values. In liver, kidney, heart, gizzard, breast, or thigh, no selenoprotein transcript was increased ≥2.0X, and no selenoprotein transcript was decreased ≤0.5X by 2 or 5 µg Se/g diet as compared to poults fed 0.4 µg Se/g diet. Of the 112 Se status biomarkers reported in this study, liver Se concentration was the only biomarker markedly altered by high Se status. This study provides evidence of no adverse effects in turkey poults fed up to 5 µg Se/g diet as inorganic Se. Thus, the FDA limit for Se supplementation in turkey feed can be safely raised to 0.5 µg Se/g diet. Future studies are needed to identify biomarkers for high Se status and to better understand how turkeys maintain Se homeostasis and resist Se toxicity.

Longitudinal selenium status in healthy British adults: assessment using biochemical and molecular biomarkers.

Human selenium (Se) requirements are currently based on biochemical markers of Se status. In rats, tissue glutathione peroxidase-1 (Gpx1) mRNA levels can be used effectively to determine Se requirements; blood Gpx1 mRNA levels decrease in Se-deficient rats, so molecular biology-based markers have potential for human nutrition assessment. To study the efficacy of molecular biology markers for assessing Se status in humans, we conducted a longitudinal study on 39 subjects (age 45 +/- 11) in Reading, UK. Diet diaries (5 day) and blood were obtained from each subject at 2, 8, 17 and 23 weeks, and plasma Se, glutathione peroxidase (Gpx3) enzyme activity, and selenoprotein mRNA levels were determined. There were no significant longitudinal effects on Se biomarkers. Se intake averaged 48 +/- 14 microg/d. Plasma Se concentrations averaged 1.13 +/- 0.16 micromol/l. Plasma Se v. energy-corrected Se intake (ng Se/kJ/d) was significantly correlated, but neither Gpx3 activity v. Se intake (ng Se/kJ/d) nor Gpx3 activity v. plasma Se was significantly correlated. Collectively, this indicates that subjects were on the plateaus of the response curves. Selenoprotein mRNAs were quantitated in total RNA isolated from whole blood, but mRNA levels for Gpx1, selenoprotein H, and selenoprotein W (all highly regulated by Se in rodents), as well selenoprotein P, Gpx3, and phospholipid hydroperoxide glutathione peroxidase were also not significantly correlated with plasma Se. Thus selenoprotein molecular biomarkers, as well as traditional biochemical markers, are unable to further distinguish differences in Se status in these Se replete subjects. The efficacy of molecular biomarkers to detect Se deficiency needs to be tested in Se-deficient populations.

Selenium regulation of selenoprotein enzyme activity and transcripts in a pilot study with Founder strains from the Collaborative Cross.

Rodents and humans have 24-25 selenoproteins, and these proteins contain the 21st amino acid, selenocysteine, incorporated co-translationally into the peptide backbone in a series of reactions dependent on at least 6 unique gene products. In selenium (Se) deficiency, there is differential regulation of selenoprotein expression, whereby levels of some selenoproteins and their transcripts decrease dramatically in Se deficiency, but other selenoprotein transcripts are spared this decrease; the underlying mechanism, however, is not fully understood. To begin explore the genetic basis for this variation in regulation by Se status in a pilot study, we fed Se-deficient or Se-adequate diets (0.005 or 0.2 µg Se/g, respectively) for eight weeks to the eight Founder strains of the Collaborative Cross. We found rather uniform expression of selenoenzyme activity for glutathione peroxidase (Gpx) 3 in plasma, Gpx1 in red blood cells, and Gpx1, Gpx4, and thioredoxin reductase in liver. In Founder mice, Se deficiency decreased each of these activities to a similar extent. Regulation of selenoprotein transcript expression by Se status was also globally retained intact, with dramatic down-regulation of Gpx1, Selenow, and Selenoh transcripts in all 8 strains of Founder mice. These results indicate that differential regulation of selenoprotein expression by Se status is an essential aspect of Se metabolism and selenoprotein function. A few lone differences in Se regulation were observed for individual selenoproteins in this pilot study, but these differences did not single-out one strain or one selenoprotein that consistently had unique Se regulation of selenoprotein expression. These differences should be affirmed in larger studies; use of the Diversity Outbred and Collaborative Cross strains may help to better define the functions of these selenoproteins.

Impact of Glutathione Peroxidase-1 (Gpx1) Genotype on Selenoenzyme and Transcript Expression When Repleting Selenium-Deficient Mice.

Glutathione peroxidase (Gpx1) is the major selenoprotein in most tissues in animals. Knockout (KO) of Gpx1 decreases Gpx1 activity to near zero and substantially reduces liver selenium (Se) levels, but has no overt effects in otherwise healthy mice. To investigate the impact of deletion of Gpx1 on Se metabolism, Se flux, and apparent Se requirements, KO, Gpx1 heterozygous (Het), and Gpx1 wild-type (WT) mice were fed Se-deficient diet for 17 weeks, then repleted with graded levels of Se (0-0.3 µg Se/g as Na2SeO3) for 7 days, and selenoprotein activities and transcripts were determined in blood, liver, and kidney. Se deficiency decreased the activities of plasma Gpx3, liver Gpx1, liver Txnrd, and liver Gpx4 to 3, 0.3, 11, and 50% of WT Se-adequate levels, respectively, but the Gpx1 genotype had no effect on growth or changes in activity or expression of selenoproteins other than Gpx1. Se repletion increased selenoprotein transcripts to Se-adequate levels after 7 days; Se response curves and apparent Se requirements for selenoprotein transcripts were similar to those observed in studies starting with Se-adequate mice. With short-term Se repletion, selenoenzyme activities resulted in three Se response curve patterns: (1) liver and kidney Gpx1, Gpx4, and Txnrd activities were sigmoidal or hyperbolic with breakpoints (0.08-0.19 µg Se/g) that were double those observed in studies starting with Se-adequate mice; (2) red blood cell Gpx1 activity was not significantly changed; and (3) plasma Gpx3 activity only increased substantially with 0.3 µg Se/g. Plasma Gpx3 is secreted from kidney. In this short-term study, kidney Gpx3 mRNA reached plateau levels at 0.1 µg Se/g, and other kidney selenoenzyme activities reached plateau levels at ≤ 0.2 µg Se/g, so sufficient Se should have been present in kidney. Thus, the delayed increase in plasma Gpx3 activity suggests that newly synthesized and secreted kidney Gpx3 is preferentially retained in kidney or rapidly cleared by binding to basement membranes in kidney or in other tissues. This repletion study shows that loss of capacity to incorporate Se into Gpx1 in Gpx1 KO mice does not dramatically alter expression of other Se biomarkers, nor the short-term flux of Se from intestine to liver to kidney.