Journal of Nutrition EB Program 2010 Abstracts

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yeh, J.-Y.
Right arrow Articles by Whanger, P. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yeh, J.-Y.
Right arrow Articles by Whanger, P. D.

The Journal of Nutrition Vol. 127 No. 11 November 1997, pp. 2165-2172
Copyright ©1997 by the American Society for Nutritional Sciences

Dietary Selenium Increases Selenoprotein W Levels in Rat Tissues1,2

Jan-Ying Yeh, Susan C. Vendeland, Qiu-ping Gu, Judy A. Butler, Bor-Rung Ou3, and Philip D. Whanger

Departments of Agricultural Chemistry and Animal Sciences, Oregon State University, Corvallis, OR 97331

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED

ABSTRACT

Two experiments were conducted to evaluate the influence of dietary selenium (Se) on tissue levels of selenoprotein W (Se-W) in rats. Se dependent glutathione peroxidase (GPX) activity and Se levels were also determined for comparative measurements. In the first experiment, rats were fed a basal diet deficient in Se or supplemented with either 0.1 or 4.0 mg Se (as selenite) per kg diet for 6 wk. Se-W levels were significantly higher in muscle, spleen and testes of rats fed 0.1 mg Se per kg diet compared to those fed the deficient diet (controls), and those fed 4.0 mg Se per kg diet had significantly higher levels in muscle, brain and spleen (P < 0.05) than those fed 0.1 mg Se per kg diet. No further increases, however, occurred in the tests. There was a significant increase (P < 0.05) of mRNA encoding Se-W in muscle with each increase of dietary Se. In the second experiment rats were fed the basal diet or this diet plus 0.01, 0.03, 0.06, 0.1, 1.0, 2.0 or 4.0 mg Se per kg diet. The levels of Se-W in muscle did not increase (P < 0.05) until 0.06 mg Se per kg diet were fed to rats. A very marked increase (P < 0.05) occurred when 1.0 mg Se per kg diet was fed with no further increases with higher levels. There was a linear increase of Se-W in brain (r = 0.89) and spleen (r = 0.98) with the Se concentration in the diet up to 0.1 mg Se per kg where a plateau was reached. The testes showed a different pattern in that a very marked increase (P < 0.01) occurred when only 0.01 mg Se per kg diet was fed where an inflection was reached. Except for muscle, GPX activities reached a plateau in all tissues when diets containing 0.06 to 0.1 mg supplemental Se per kg were fed. The Se concentration in these tissues increased at a linear rate with the Se concentration in the diets up to 0.1 mg Se per kg where it continued to rise at a different rate. The results indicate that in rats, the regulation of Se-W by Se is different for various tissues and differs from that for GPX.

KEY WORDS: selenoprotein W · glutathione peroxidase · selenium rats · muscle · brain · testes · spleen

INTRODUCTION

At present there are four known members of the family of glutathione peroxidases (GPX).4 The first to be recognized was the cellular GPX from rats (GPX1, Rotruck et al. 1973). Subsequently, a GPX was purified from human plasma (GPX3) and shown to be immunochemically distinct from the GPX1 (Takahashi et al. 1987). A third GPX (GPX4) was discovered to be capable of reducing fatty acid hydroperoxides esterified to phospholipids (Ursini et al. 1985). Evidence was obtained for a fourth GPX (GPX2) found predominantly in the gastrointestinal tract (Chu et al. 1993). The use of graded levels of dietary Se allows investigators to determine the Se response curves for several variables. GPX4 and GPX1 have been shown to be differentially regulated by Se status (Lei et al. 1995). Liver GPX1 activity was highly regulated by Se status with virtually no activity in rats fed a Se deficient diet. Relative plateau levels of activity were reached with 0.1 mg Se per kg diet (Hafeman et al. 1974, Knight and Sunde 1987). In contrast, liver GPXs4 was reduced by only 40% with the deficient diet but reached relative plateau levels with dietary Se levels less than 0.1 mg Se per kg (Lei et al. 1995). Therefore, since the two GPXs are differentially regulated, other selenoproteins are likely to be affected differently by Se.

A low molecular weight selenoprotein, called selenoprotein W (Se-W), was recently purified and characterized (Vendeland et al. 1993). In rats fed a nonpurified diet, Western blots indicated that this selenoprotein is present in skeletal muscle, brain, spleen and testes (Yeh et al. 1995). This selenoprotein was undetectable in skeletal muscle of deficient rats, suggesting that Se status affects the level of this selenoprotein. This is supported by information showing that mRNA levels for Se-W in skeletal muscle are affected by Se status (Vendeland et al. 1995). However, no detailed information is available on the regulation of this selenoprotein in tissues by Se and this is the purpose of the present communication. Two experiments were conducted. In the first, the influence of three dietary levels of Se on Se-W in tissues was investigated and the mRNAs for this selenoprotein determined in nine tissues from rats fed the highest (4 mg/kg) amount of Se and in the muscle from rats fed all three diets. In the second experiment, the influence of nine concentrations of dietary Se on Se-W concentration was investigated in muscle, brain, testis and spleen, but no measurements on mRNA levels were made.

MATERIALS AND METHODS

Animal treatment. This research was reviewed and approved by the animal care committee on the Oregon State University campus. Sprague Dawley rats, purchased from Simonsen Laboratories, Gilroy, CA, were used in both experiments. All rats had free access to food and water, and a record of food consumption was kept. In the first experiment, male weanling rats (60 ± 5 g) were fed (5/diet) either the deficient diet (no Se added) or this diet supplemented with either 0.1 or 4.0 mg Se/kg for 6 wk. In the second experiment, weanling rats (58 ± 6 g) were fed (5/diet) the deficient diet or this diet supplemented with either 0.01, 0.03, 0.06, 0.1, 1.0, 2.0 and 4.0 mg Se/kg for 8 wk. Se was added in the form of sodium selenite. The basal diet has been described (Butler et al. 1989) and was composed of (g/kg) 300 torula yeast (Rhinelander Paper Co., Rhinelander, WI), 510 sucrose, 90 purified cellulose (Solka Floc, Brown Co., Berlin, NH) 50 corn oil, 35 AIN-93M mineral mix without Se and 10 AIN-76 vitamin mix (American Institute of Nutrition 1977), plus 3 L-methionine and 2 choline citrate. The basal diet in both experiments was shown by analysis to contain about 4 µg Se per kg diet. In both studies, the rats were anesthetized with sodium pentobarbital (80 mg/kg i.p.), and blood was taken via cardiac puncture. After the rats were decapitated while under anesthesia, the tissues were removed and frozen immediately at -70°C. The tissues were kept frozen at this temperature until used. Se-W levels, GPX activities and Se were measured in all tissues examined.

Western blot analysis. A portion of each tissue was homogenized in a Tris buffer, centrifuged to obtain supernatants for Western blots as described previously (Yeh et al. 1995 and 1997a). Briefly, this involves the following procedures: protein content was measured in the supernants by the dye-binding assay of Bradford (1976) using bovine serum albumin (Bio-Rad, Richmond, CA) as standard. Samples (200 µg protein each) were electrophoretically separated on SDS-polyacrylamide 7.5 to 15% gradient gels as described by Laemmli (1970). Proteins were transferred onto nitrocellulose membranes according to the method of Tobin et al. (1979). These membranes were incubated with rabbit anti-Se-W polyclonal (Yeh et al. 1995) antibody followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad), washed, and the specific binding of anti Se-W antibody was detected with ECL detection system (Amersham, Arlington Heights, IL) as described (Yeh et al. 1997b). The membranes were then exposed to Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY) for periods below the saturation range as determined by multiple exposure times. Two or more films were exposed at once, stacked on top of one another for this procedure. Developed films were scanned with a Personal Densitometer SI and analyzed with the ImageQuant program (both from Molecular Dynamics, Sunnyvale, CA).

Total RNA extraction. Extraction of total RNA has been described previously (Chomczynski and Sacchi 1987) with the modifications noted earlier (Yeh et al. 1997b). The precipitated RNA pellets were resuspended, washed twice with 70% ethanol and dried under a vacuum. The dried RNA pellets were then dissolved in diethylpyrocarbonate (DEPC)-treated water and quantitated by spectrophotometry.

Northern blot analysis. Northern blots were performed on total RNA from testis, lungs, muscle, spleen, liver, kidney, intestine, brain and heart from rats fed the diet with 4.0 mg Se per kg, and from muscle of rats fed all three diets in the first experiment. Because of the results of the first experiment, Northern blots were not performed on any of the tissue preparations from rats of the second experiment. Northern blot analysis (20 µg RNA) was conducted as previously described (Yeh et al. 1997b). Se-W cDNA and 18S ribosomal RNA oligonucleotides (Giovannoni 1991) were labeled using DIG Labeling Kit (Boehringer Mannheim, Indianapolis, IN). Briefly, membranes were prehybridized then heat-denatured DIG labeled Se-W probe was added and hybridized overnight. The membranes were washed, and anti-digoxigenin-alkaline phosphatase conjugated antibody was incubated with membranes. Unbound antibody was removed by washing and the membranes were equilibrated and exposed to substrate. The membranes were exposed to Kodak X-OMAT film (Eastman Kodak Co.) for periods of time below the saturation point. Developed films were scanned with a Personal Densitometer SI and analyzed by the ImageQuant program (Molecular Dynamics). Before hybridization to the internal control probe, the membranes were rinsed thoroughly in water and incubated in stripping buffer. After rinsing with water, the membranes were used for hybridization of internal control probe (Krueger and Williams 1995).

Glutathione peroxidase activity and Se content. GPX1 activity was measured by an enzyme-coupled method using glutathione reductase with hydrogen peroxide (0.171 mmol/L) as substrate (Paglia and Valentine 1967). After digestion of the tissue fractions with nitric and perchloric acids, Se was determined by the semiautomated fluorimetric method (Brown and Watkinson 1977) using an Alpkem II system (Alpkem Corp., Milwaukie, OR).

Statistical analysis. Data were examined for equal variance and normal distribution prior to statistical analysis. Where necessary to achieve homogeneity, the data were subjected to logarithmic transformations. Mean values were compared by analysis of variance (ANOVA) with Fisher's least-significant difference (LSD) method for comparing groups (Steel and Torrie 1980). In the first experiment, only GPX1 activity in the testis was logarithmically transformed before statistical analysis was done. In the second experiment, data for Se-W content, GPX1 activity and Se concentrations in spleen and testis; Se-W content and Se concentration in brain; GPX1 activity in muscle; and liver Se were logarithmically transformed before statistical analyses were done. Regression analysis was done on some of the data (blood and liver Se, GPX1, Se and Se-W in muscle, brain, spleen and testis with dietary Se in Experiment 2). A significance level of P <= 0.05 was adopted for all comparisons.

RESULTS

Experiment 1. There were no differences in the food consumption of the rats in this study. However, the rats fed the deficient diet gained (181 ± 5 g) significantly less (P < 0.05) than those fed the other two diets (197 ± 5 g for those fed 0.1 mg Se per kg diet or 211 ± 4 g for those fed 4.0 mg Se per kg diet).

Except for the brain and the testes from rats fed 0.1 and 4.0 mg Se per kg diet, there was a corresponding increase of tissue Se with each increase of dietary Se (Table 1). Except for the brain, the Se concentration was higher in tissues from rats fed the 0.1 mg Se per kg diet than those fed the basal diet, and except for testis and brain those fed the 4.0 mg Se per kg diet were higher than in tissues from those fed the 0.1 mg Se per kg diet.

Table 1. Tissue Se concentration of rats fed Torula yeast diets supplemented with 0, 0.1 or 4.0 mg Se as selenite per kg1

[View Table]

There was a corresponding significant increase (P < 0.05) in the GPX1 (GPX3 in plasma) activities with each increase of dietary Se in plasma, testes and spleen (Table 2). However, even though this activity was higher in muscle in rats fed the 0.1 mg Se per kg diet than muscle from deficient rats, there was no further significant increase when 4.0 mg Se was present in the diet. The brain showed a different pattern in that this activity was not significantly higher in rats fed the 0.1 mg Se diet versus those fed the basal diet, but was significantly higher (P < 0.05) in rats fed the 4.0 mg Se per kg diet.

Table 2. Glutathione peroxidase activities of rats fed Torula yeast diets supplemented with 0, 0.1 or 4 mg Se as selenite per kg1

[View Table]

Western blots were used to determine the content of Se-W in brain, testis, muscle and spleen from the densities of the bands (Table 3). Se-W was not detectable in muscle of deficient rats, but was present in muscle of rats fed the diet with 0.1 mg Se per kg. Those fed 4.0 mg Se per kg had significantly higher (P < 0.05) levels in the muscle than in muscle from those fed 0.1 mg Se per kg diet. The other tissues showed different patterns. Se-W in spleen increased with each increase of dietary Se, whereas the content in brain was the same in rats fed the deficient diet and the diet with 0.1 mg Se per kg, but higher in rats fed the diet with 4.0 mg Se per kg. In contrast, Se-W in testis was significantly higher from rats fed diet with 0.1 mg Se per kg than the deficient rats with no further increase with higher Se intake.

Table 3. Effects of Se status on selenoprotein W of rats fed Torula yeast diets supplemented with 0, 0.1 or 4 mg Se as selenite per kg1

[View Table]

Northern blots of tissues from rats fed the diet with 4.0 mg Se per kg are shown in Figure 1. Of the tissues examined, Se-W mRNAs were found only in muscle, brain, testis and spleen even though high dietary levels of Se were fed. The intensities of the bands were greatest for the muscle and brain. This information was used to assess the relative levels of mRNA in muscle.

Fig. 1. Tissue distribution of selenoprotein W mRNA from a rat fed 4 mg Se per kg diet. A: Hybridization to Se-W cDNA probe. B: Hybridization to the internal control probe (18S rRNA). T, testis; Lu, lung; M, muscle; Sp, spleen; Li, liver; K, kidney; I, intestine; B, brain; H, heart.
[View Larger Version of this Image (123K GIF file)]

The Northern blots of the muscle from rats fed the three levels of Se are shown in Figure 2. The intensities of the bands increased with each increase of the dietary Se levels. The quantitated band densities of the Northern blots were used to determine the relative Se-W mRNA levels in muscle of rats fed the three levels of dietary Se (Table 4). The mRNA levels when expressed as the ratio to the internal standard increased significantly (P < 0.05) with each increase of dietary Se.

Fig. 2. Northern blots of selenoprotein W mRNA in skeletal muscles from rats fed Torula yeast diets with additions of 0, 0.1 and 4.0 mg Se per kg. A: Hybridization to Se-W cDNA probe. B: Hybridization to internal control probe (18S rRNA). Lanes 1-6 are from rats fed basal diet. Lanes 7-11 are from rats fed 0.1 mg Se per kg diet. Lanes 12-15 are from rats fed 4.0 mg Se per kg diet.
[View Larger Version of this Image (131K GIF file)]

Table 4. Northern blot data of selenoprotein W mRNA and internal control (18S rRNA) mRNA in muscles of rats fed Torula yeast diets supplemented with 0, 0.1 and 4 mg Se as selenite per kg1

[View Table]

Experiment 2. There were no differences in feed consumption between the different dietary groups (data not shown). However, there were some differences in weight gain (g/56 d). Those fed the diet with 0.03 mg Se per kg gained (231 ± 5 g), significantly (P < 0.05) more than rats fed the diets with the two lower levels (0 Se, 196 ± 5 g; 0.01 Se, 207 ± 7 g) of this element. The weight gains of rats fed 0.03, 0.06 (207 ± 4 g), 0.1 (220 ± 7 g), 1 (216 ± 4 g) and 2 (211 ± 10 g) mg Se/kg diet were not significantly different. The weight gains (196 ± 7 g) of those rats fed the diet with 4 mg Se per kg were not significantly different from rats fed either the diet with no added Se or 0.01 mg Se per kg.

The whole blood and hepatic Se levels were determined as a measure of the Se status of the animals (Figure 3). Significant correlations of Se concentration occurred in both whole blood (r = 0.88) and liver (r = 0.96) with the dietary levels of this element. There was a linear increase up to 0.1 mg Se per kg diet where an inflection occurred with higher Se intakes (Figure 3). The blood Se concentrations were significantly higher as compared to the previous level fed with all levels of Se fed to the rats. Except for rats fed 0.06 and 0.1 mg Se per kg diet, similar results were obtained for the livers (Figure 3, top).

Fig. 3. Se concentration of whole blood and liver from rats fed various levels of dietary Se. More detailed patterns for 0 to 0.1 mg added Se per kg diet are shown in the top graph. Points on lines for either whole blood or liver with different letters are significantly different (P < 0.05). The correlation coefficients were 0.99 and 0.98, respectively, for liver and whole blood with dietary Se of no addition to 0.1 mg Se per kg; 0.99 and 0.99, respectively, for dietary Se between 0.1 and 4.0 mg Se per kg; and 0.96 and 0.88, respectively, for the entire dietary range.
[View Larger Version of this Image (20K GIF file)]

Western blots of Se-W in skeletal muscle from rats fed various levels of dietary Se is shown in Figure 4. This information was used to determine the relative amounts of Se-W in muscle from the experimental rats. Identical procedures were used to determine the relative amounts of Se-W in the other tissues (brain, testis and spleen) studied.

Fig. 4. Western blot analysis of selenoprotein W in skeletal muscles from rats fed various levels of dietary Se. Lanes A 1-5 are from rats fed basal diet. Lanes B 1-5 are from rats fed 0.01 mg Se per kg diet. Lanes C 1-5 are from rats fed 0.03 mg Se per kg diet. Lanes D 1-5 are from rats fed 0.06 mg Se per kg diet. Lanes E 1-5 are from rats fed 0.1 mg Se per kg diet. Lanes F 1-5 are from rats fed 1.0 mg Se per kg diet. Lanes G 1-5 are from rats fed 2.0 mg Se per kg diet. Lanes H 1-5 are from rats fed 4.0 mg Se per kg diet.
[View Larger Version of this Image (76K GIF file)]

The Se concentration, GPX1 activity and Se-W levels in muscle followed different patterns in response to Se intake (Figure 5). Significant increases of muscle Se occurred with low intake of dietary Se, but increases of GPX1 or Se-W did not occur until 0.06 mg Se per kg diet were fed as compared to rats fed the basal diet (Figure 5, top). The first detectable level of Se-W was in muscle from rats fed the diet with 0.06 mg Se per kg while very marked increases (P < 0.01) occurred with the two next highest levels of Se used. Both Se-W and GPX1 activity reached a plateau with 1.0 mg Se per kg diet, but the Se concentration continued to rise with each increase of dietary Se (Figure 5, bottom). There was a linear increase of Se concentration, GPX1 activity and Se-W content up to 0.1 mg Se per kg diet where an inflection occurred with all three biomarkers.

Fig. 5. Relative percentages of selenoprotein W, glutathione peroxidase activity and Se in skeletal muscle from rats fed various levels of dietary Se. More detailed patterns for 0 to 0.1 mg added Se per kg diet are shown in the top graph. The values for Se-W, GPX and Se for rats fed 0.1 mg Se per kg diet were set at 100 and all of the other values are expressed as a percentage of this value. The values for Se-W (scan units), GPX [nmol NADPH oxidized/(min·mg protein)] and Se concentration (nmol/g dry wt) for rats fed 0.1 mg Se per kg diet were 651 ± 63; 62 ± 6, and 4.6 ± 0.1, respectively. Points with different letters within a particular biomarker are significantly different. The correlation coefficients were 0.98, 0.88, and 0.96, respectively, for GPX, Se-W and Se with no addition to 0.1 mg Se per kg diet; 0.63, 0.74 and 0.98, respectively, for dietary Se of 0.1 to 4.0 mg Se per kg diet, and 0.77, 0.86, and 0.90, respectively, over the entire dietary range of Se.
[View Larger Version of this Image (21K GIF file)]

As compared to the muscle, different patterns for GPX1 activity, Se concentration and Se-W content occurred in brain in response to dietary Se (Figure 6). Between 0.01 and 0.1 mg Se per kg diet, there were no differences in the Se content in the brain, with significant increases above 0.1 mg Se per kg diet (Figure 6, top). This is in contrast to Se-W where an increase occurred with most increases of dietary Se. Except at the lower levels of dietary Se, GPX1 activity followed a similar pattern. All three biomarkers reached an inflection point at 0.1 mg Se per kg diet (Figure 6, bottom), with linear increases afterwards.

Fig. 6. Relative percentages of selenoprotein W level, glutathione peroxidase and Se in brain from rats fed various levels of dietary Se. More detailed patterns for 0 to 0.1 mg added Se per kg diet are shown in the top graph. The values for Se-W, GPX and Se for rats fed 0.1 mg Se per kg diet were set at 100 and all of the other values are expressed as a percentage of this value. The values for Se-W (scan units), GPX [nmol NADPH oxidized/(min·mg protein)] and Se concentration (nmol/g dry wt) for rats fed 0.1 mg Se per kg diet were, respectively: 528 ± 65; 111 ± 5, and 6.4 ± 0.3. Points with different letters within a particular biomarker are significantly different. The correlation coefficients were 0.95, 0.89 and 0.57, respectively, for GPX, Se-W and Se with no addition to 0.1 mg Se per kg diet; 0.94, 0.96 and 0.98, respectively, for dietary Se of 0.1 to 4.0 mg Se per kg, and 0.80, 0.81 and 0.98, respectively, over the entire dietary range of Se.
[View Larger Version of this Image (20K GIF file)]

The increase of Se-W in the testes with various levels of Se was different from that seen in the other tissues (Figure 7). Although there were significant increases in all three biomarkers, the greatest increase occurred with Se-W content between the rats fed the basal diet and those fed the diet with 0.01 mg Se per kg diet (Figure 7, top). Even though there were increases above 0.01 mg Se per kg diet, none of them were as great as that between the basal and 0.01 mg Se per kg diet. Increases of GPX1 activity and Se concentration occurred with most increases of dietary Se. The correlation coefficients for GPX1 activity, Se-W content and concentration of Se over the entire dietary range of Se were 0.57, 0.43 and 0.58, respectively. All biomarkers reached an inflection at 0.1 mg Se per kg diet (Figure 7, bottom), where a fairly level plateau was reached afterwards.

Fig. 7. Relative percentages of selenoprotein W level, glutathione peroxidase activity and Se in testis from rats fed various levels of dietary Se. More detailed patterns for 0 to 0.1 mg added Se per kg diet are shown in the top graph. The values for Se-W, GPX and Se for rats fed 0.1 mg Se per kg diet were set at 100 and all of the other values expressed as a percentage of this value. The values for Se-W (scan units), GPX [nmol NADPH oxidized/(min·mg protein)] and Se concentration (nmol/g dry wt) for rats fed 0.1 mg Se per kg diet were, respectively: 70 ± 2, 700 ± 52, and 8.4 ± 0.5. Points with different letters within a particular biomarker are significantly different. The correlation coefficients were 0.88, 0.58 and 0.71, respectively for GPX, Se-W and Se with no addition to 0.1 mg Se per kg diet; 0.33, 0.19 and 0.84, respectively, for dietary Se of 0.1 to 4.0 mg Se per kg; and 0.55, 0.43 and 0.58, respectively, over the entire dietary range of Se.
[View Larger Version of this Image (20K GIF file)]

A different pattern for the three biomarkers was noted in the spleen (Figure 8). Even though GPX1 activity and Se-W content reached a plateau with 0.1 mg Se per kg diet, the Se concentration continued to rise. Similar to the patterns in other tissues, all three biomarkers reached an inflection at 0.1 mg Se per kg diet (Figure 8, bottom). The correlation coefficients, respectively, for GPX1 activity, Se-W content and Se concentration over the entire dietary range were 0.75, 0.46 and 0.97. 

Fig. 8. Relative percentages of selenoprotein W, glutathione peroxidase activity, and Se in spleen from rats fed various levels of dietary Se. More detailed patterns for 0 to 0.1 mg added Se per kg diet are shown in the top graph. The values for Se-W, GPX and Se for rats fed 0.1 mg Se per kg diet were set at 100 and all of the other values are expressed as a percentage of this. The values for Se-W (scan units), GPX [nmol NADPH oxidized/(min·mg protein)] and Se concentration (nmol/g dry wt) for rats fed 0.1 mg Se per kg diet were, respectively: 1805 ± 102, 528 ± 48 and 18.1 ± 1.2. Points with different letters within a particular biomarker are significantly different. The correlation coefficients were 0.96, 0.98 and 0.97, respectively, for GPX, Se-W and Se with no addition to 0.1 mg Se per kg diet; 0.99, 0.65, and 0.98, respectively, for dietary Se of 0.1 to 4.0 mg Se per kg; and 0.75, 0.46 and 0.97, respectively, over the entire dietary range of Se.
[View Larger Version of this Image (20K GIF file)]

DISCUSSION

The results of feeding various levels of dietary Se indicated that this element affects Se-W differently in the tissues studied. The data from first experiment indicated that Se-W responded to Se intake, but those of the second experiment gave more precise information on the regulation of Se-W by Se. It is obvious that the regulation of Se-W by Se is different among tissues and also differs from that for GPX1. A very marked increase of Se-W was found in muscle when levels of Se greater than 0.06 mg Se per kg were fed to the rats (Figures 4 and 5). This is in contrast to the testes where a marked increase occurred with only 0.01 mg Se per kg diet (Figure 7). The stability of mRNA is probably regulated through the interaction of cis-elements with RNA-binding proteins that can affect the susceptibility of RNA to nucleolytic attack. A well-characterized example is the iron-responsive element (IRE) present in the 3' untranslated region of transferrin receptor mRNA (Casey et al. 1988). The IRE forms a stem-loop structure that is recognized by an IRE-binding protein. Se-W mRNA also contains a stem-loop structure in the 3' untranslated region analogous to the conserved selenocysteine insertion sequence (SECIS) element in other mammalian selenoprotein mRNAs (Gu et al. 1997). The SECIS element is considered to be an essential structure for selenocysteine incorporation at the UGA codon (Berry et al. 1994, Walczak et al. 1996). It is possible that the same stem-loop structure has a dual function involved in selenocysteine insertion as well as selenoprotein mRNA stabilization. It is postulated that there are Se responsive turnover elements and that they are more sensitive to Se in the testes than the muscle. The patterns in the brain (Figure 6) and spleen (Figure 8) were different from either the muscle or testis.

The regulation of GPX1 activity by Se is different from Se-W in the tissues studied. The plateau for GPX1 was reached at 0.1 mg Se per kg diet in the brain (Figure 6), at 0.06 mg Se per kg diet in both the testes (Figure 7) and spleen (Figure 8) but further increases occurred in muscle when 1.0 and 2.0 mg Se per kg diet were fed (Figures 4 and 5). As far as we know, these results with skeletal muscle for GPX1 have not been reported previously. The regulation of GPX1 and GPX4 activities were studied in liver, heart, kidney, lung and testes with rats (Lei et al. 1995). Plateaus in activities for both isoenzymes of GPX was reached from 0.075 to 0.130 mg Se per kg diet. Except for the muscle, the present results with the various tissues are consistent with those obtained by other investigators who indicated this plateau is reached around 0.1 mg Se per kg diet (Hafeman et al. 1974, Knight and Sunde 1987).

The rapid response of Se-W to low levels of Se in the testes is similar to that reported for GPX4. The levels of Se-W increased very markedly with only 0.01 mg Se per kg (Figure 7) as compared to a reported plateau for GPX4 at 0.025 mg Se per kg in the testes (Lei et al. 1995). This may suggest that these two selenoproteins have similar response elements in testes.

The question arises as to the mechanism of the regulation of Se-W by Se. Table 4 indicates that the mRNA levels for selenoproteins are sensitive to Se. However, additional information is needed to evaluate this regulation. In other work, nuclear run-off transcription assays showed that Se status has no effect on the transcription of GPX1 mRNA levels (Christensen and Burgener 1992, Toyoda et al. 1990), suggesting that Se status influences the stability of GPX1 mRNA. Unpublished work from our laboratory also indicates that Se affects the stability of mRNA for Se-W instead of transcription. The reason for different responses between GPX1 and Se-W in various tissues are not known. One hypothesis could be that there are more rapid-turnover elements in GPX1 than in Se-W mRNA. Reduced Se status would elicit degradation of GPX1 mRNA but not in Se-W mRNA or mRNA for other selenoenzymes. An alternative hypothesis is that SECIS in the mRNA, which are necessary for Se insertion (Berry et al. 1993), may also affect stability. For example, Berry et al. (1993) found different levels of deiodinase activity in cells transfected with recombinant deiodinase fusion genes, depending on which selenoprotein provided the SECIS.

Prior work indicated that Se-W was present in the muscle, brain, testes and spleen of rats (Yeh et al. 1995) but not in the heart (Figure 1). However, there are some species differences in the tissue distribution of this selenoprotein. When Se is given to sheep, the Se-W content is highest in the heart and muscle (Yeh et al. 1997a). This is interesting because these are the tissues which are affected in Se deficient sheep (Schubert et al. 1961) and could be the reason there are lesions in the heart from deficient sheep but not in this organ of rats.

There is some information to suggest different depletion rates of various tissues for Se-W. When sheep were fed a Se deficient diet for about three months, Se-W was significantly lower in all tissues examined except for the brain in comparison to Se supplemented sheep (Yeh et al. 1997a). However, the Se content in the brain was 50% lower and the GPX1 activity was 30% lower in brain of deficient sheep versus those receiving Se. This suggests preferential retention of Se-W in the brain as compared to Se or GPX1.

The metabolic function of Se-W is not known. Selenoprotein P is the other most studied selenoprotein without a known function. Selenoprotein P is present in plasma of rats (Read et al. 1990) and humans (Deagen et al. 1993) and has been shown to contain 10 selenocysteines (Hill et al. 1991). Like Se-W, the levels of selenoprotein P are affected by the Se status of the animal (Yang et al. 1989).

Most studies do not report a decreased weight gain in rats fed Se deficient diets but this is probably because the Se concentration was not low enough to produce an effect. We were fortunate to obtain torula yeast with extremely low levels of Se in which the basal diet contained only 4 ng Se per g diet. The growth response to Se additions is consistent with work of other investigators when Se was added to extremely low diets for rats (Hafeman et al. 1974, Lei et al. 1995). Experiment 1 was conducted for 6 wk whereas Experiment 2 was not terminated until after 8 wk. In comparison to those fed 0.1 mg Se per kg diet, no difference in weight gain was noted in rats fed 4.0 mg Se per kg diet in the first experiment, but a decrease was noted in Experiment 2. Presumably, the additional 2 wk in the second experiment accounted for this difference.

Se deficiency results in a muscular and cardiac degeneration, white muscle disease, in lambs and calves (Schubert et al. 1961). Likewise, a severe nutritional Se deficiency in discrete regions of China was associated with an endemic juvenile cardiomyopathy disorder called Keshan disease (Chen et al. 1980). Muscle weakness has been shown to be prevented by Se supplementation in patients on long-term parenteral nutrition (Brown et al. 1986). Therefore, there is sufficient evidence to indicate that Se is important for normal muscle metabolism. Whether Se-W has any significant role in muscle metabolism cannot be determined from the present work, but it is predicted that the higher level in the muscle is not a coincidence.

ACKNOWLEDGMENTS

The technical assistance of Azizah Mohd is greatly appreciated. The assistance of Calvin Nunn with the statistical analysis is acknowledged.

FOOTNOTES

1   Published with the approval of the Oregon State Agricultural Experiment Station as Technical Paper 10,688. This research was supported by Public Health Service Research Grant DK 38306 from the National Institute of Diabetes and Digestive and Kidney Diseases.
2   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3   Present address: Department of Animal Science, Tunghai University, Taichung, Taiwan, 407, Republic of China.
4   Abbreviations used: GPX, glutathione peroxidase; GPX1, cellular GPX (GSH2:H2O oxidoreductase, EC 1.11.1.9); GPX3, plasma GPX; GPX4, phospholipid hydroperoxide GPX; and GPX2, GPX in intestinal tract.

Manuscript received 27 January 1997. Initial reviews completed 10 March 1997. Revision accepted 6 August 1997.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
R. A. Sunde, K. M. Thompson, J. K. Evenson, and B. M. Thompson
Blood Glutathione Peroxidase-1 mRNA Levels Can Be Used as Molecular Biomarkers to Determine Dietary Selenium Requirements in Rats
Experimental Biology and Medicine, November 1, 2009; 234(11): 1271 - 1279.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
K. Ashton, L. Hooper, L. J Harvey, R. Hurst, A. Casgrain, and S. J Fairweather-Tait
Methods of assessment of selenium status in humans: a systematic review
Am. J. Clinical Nutrition, June 1, 2009; 89(6): 2025S - 2039S.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
K. M. Barnes, J. K. Evenson, A. M. Raines, and R. A. Sunde
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
J. Nutr., February 1, 2009; 139(2): 199 - 206.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
X.-L. Wang
Assessment of biomarker selection in selenium-deficiency disease
Am. J. Clinical Nutrition, February 1, 2006; 83(2): 389 - 389.
[Full Text] [PDF]

Home page
 J. Nutr.Home page
W.-H. Cheng, B. A. Valentine, and X. G. Lei
High Levels of Dietary Vitamin E Do Not Replace Cellular Glutathione Peroxidase in Protecting Mice from Acute Oxidative Stress
J. Nutr., November 1, 1999; 129(11): 1951 - 1957.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yeh, J.-Y.
Right arrow Articles by Whanger, P. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yeh, J.-Y.
Right arrow Articles by Whanger, P. D.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Copyright © 1997 by American Society for Nutrition