From the Division of Gastroenterology, Department of
Medicine, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232 and § Eccles Institute of Human Genetics,
The University of Utah, Salt Lake City, Utah 84112
Received for publication, January 23, 2003, and in revised form, February 4, 2003
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ABSTRACT |
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Selenoprotein P (Se-P) contains most of the
selenium in plasma. Its function is not known. Mice with the Se-P gene
deleted (Sepp Se-P1 is the most
extreme example of the interesting protein class known as
selenoproteins. Selenoproteins contain selenocysteine in their primary
structures (1). Insertion of this 21st amino acid involves an expansion
of the genetic code by redefinition of specific internal UGA stop
codons (2). In the majority of selenoproteins there is a single
selenocysteine that is at the active site of an enzyme, and it plays a
pivotal role in a redox reaction. By contrast, Se-P has many
selenocysteines, ranging from 10 in mice and humans to 17 in zebrafish
(3, 4). The function of Se-P is not known, and there is no direct
evidence that its selenocysteine residues play a chemically active role.
Se-P is an extracellular glycoprotein that contains most of the
selenium in plasma (5). Its abundance is such that Se-P in rat plasma
accounts for 8% of the selenium in the animal. The liver is the
primary source of plasma Se-P (6), although virtually all tissues
express it. Substantial amounts of Se-P are synthesized, because the
plasma half-life of its selenium is only 3-4 h (7). This indicates
that selenium cycles through plasma Se-P at a high rate.
Because of its plasma location, Se-P has been postulated to be a
selenium transport protein (8). The selenium in Se-P is present in its
primary structure as selenocysteine residues, and so delivery of its
selenium to a cell would require degradation of the Se-P and catabolism
of its selenocysteine. That would make a selenium transport role for
the protein costly to the animal. Nevertheless, evidence suggesting a
transport function has been presented (7). Other studies have shown
that Se-P is a preferred source of selenium for Jurkat cells (9) and
embryonic neurons (10).
Another hypothesis of Se-P function is that it protects against
oxidative injury (11). Its presence has been correlated with protection
against hepatic sinusoidal endothelial cell injury by diquat in the rat
(12). Moreover, it binds to endothelial cells throughout the animal,
presumably through its heparin binding properties (13, 14).
To facilitate studies on Se-P function we have produced mice that lack
Se-P using homologous recombination. We report some of the
characteristics of these animals here, along with results that support
a selenium transport or distribution function for Se-P.
Reagents--
Restriction enzymes and ligases were obtained from
Promega (Madison, WI), New England Biolabs (Beverly, MA), and MBI
Fermentas (Amherst, NY). Cloning vectors, pBluescript and pBC, were
obtained from Stratagene (La Jolla, CA). The loxP-flanked Neo®
gene, pKT1LoxA, (15), and the TK2 gene (16) were generous gifts of
Dr. Kirk R. Thomas, University of Utah. Oligonucleotides were
synthesized by core laboratory facilities at Vanderbilt University
Medical Center and the University of Utah. [32P]dATP and
[32P]dCTP were obtained from PerkinElmer Life
Sciences. [75Se]Selenite (specific activity, 800 mCi/mg selenium) was obtained from the University of Missouri Research
Reactor Facility, Columbia, MO. NADPH was purchased from United
States Biochemical Corp. (Cleveland, OH). Glutathione reductase was
purchased from Sigma. All other chemicals were of reagent grade.
Selenoprotein P Genomic Clone Selection and Sequence--
A P1
plasmid-containing mouse genomic DNA for Sepp was purchased
from Genome Systems, Inc. (St. Louis, MO). PCR primers MG5 (5'-GATTTGTGCAAACATGGAGAAATC-3') and MG7
(5'-GAATGTAAGAGTAGGAAGACAAAG-3') were supplied to Genome Systems and
used to screen their murine 129/Sv P1 library. One clone, MG6138, was
shown to contain Sepp by Southern analysis. P1-DNA, prepared
using a procedure from Qiagen (Chatsworth, CA), was digested with
EcoRI, HindIII, or BamHI. Southern
analysis of the digested MG6138 DNA, using
[32P]dCTP-labeled 16C1 cDNA (17), showed two
HindIII fragments (approximate sizes, 6 and 2 kb), a single EcoRI fragment (10 kb), and a single
BamHI fragment (10 kb). The MG6138-HindIII digest was subcloned into pBluescript II KS. Subclones were selected by
screening with [32P]dCTP-labeled 16C1 cDNA.
Double-stranded DNA prepared from these clones was sequenced by the
Sanger dideoxy termination method.
Intron-exon boundaries were identified by a comparison of genomic DNA
sequences with the sequence of a mouse selenoprotein P cDNA
(MB23A). MB23A was obtained by screening a mouse brain cDNA library
with the HindIII fragment of MG6138. The cDNA library was a gift from Dr. Thomas Quertermous, Vanderbilt University.
Construction of Knockout Vectors--
MG6138P1 DNA was prepared
and digested with BamHI. The fragments from the digest were
ligated into pBluescript II and transformed into Escherichia
coli. Bacterial colonies were screened by PCR using primers
SeP4342 (5'-CGCAGAACGCACAAAGAATGTAGATGGC-3') and SePBsuR
(5'-GTACCCTTAGGCCAGAAGAGGGCACTGGG-3'). One clone (P1#10) selected was determined to contain a 12-kb fragment of genomic DNA
suitable for construction of a Sepp knockout vector (SePCKNT).
The SePCKNT vector was constructed using the following steps (Fig. 1).
1) The Neo® cassette, KT1LoxA, was passed through E. coli
DM1 strain to demethylate the XbaI site and then was
digested with XbaI. KT1LoxA was then inserted at the
AvrII site in P1#10, producing Sep#10Neo®. 2) SePCKN-1 was
prepared by ligation of a SalI/KpnI 7.1-kb
fragment of SeP#10Neo®, a KpnI/NotI 6.3-kb fragment of P1#10, and a SalI/NotI 3.4-kb
fragment of pBC. 3) SePCKNT was prepared by ligation of a
BamHI/NotI 2.9-kb fragment of pBluescript, a
SalI/BamHI 13.4-kb fragment of SePCKN-1, and a
NotI/XhoI 2.3-kb fragment of TK-2. SePCKNT had a
size of 18.6 kb. The total homology with Sepp genomic DNA
was 12 kb. The loxP sites within the Neo® cassette gave translational
stops in all three reading frames.
Generation of Sepp Animal Husbandry--
Adult Sepp+/
For experiments in which different amounts of selenium were fed, a
Torula yeast-based diet was used (20). The basal form of
this experimental diet contained <0.02 mg of selenium/kg. Sodium selenate was added to this diet during mixing to give final added selenium concentrations that ranged from 0.05 to 2 mg/kg. Male weanling
mice were fed basal or selenium-supplemented diet for 8 weeks. They
were weighed weekly, and mice that had lost 20% of their highest body
weight were euthanized. At 8 weeks the mice were anesthetized with
isoflurane, and blood was removed from the inferior vena cava. The
blood was treated with Na2EDTA (1 mg/ml) to prevent
coagulation, and plasma was separated by centrifugation. Liver, kidney,
heart, testis, and brain were harvested and frozen immediately in
liquid nitrogen. Plasma and tissues were stored at Biochemical Measurements--
Tissue homogenates (10%) were
prepared in 0.1 M potassium phosphate, pH 7.5. Supernatants
from centrifugation of the tissue homogenates at 13,000 × g for 30 min were used for measurement of glutathione
peroxidase activity. The coupled method was used with 0.25 mM hydrogen peroxide as substrate (21). Plasma Se-P was
measured with a radioimmunoassay (22) that utilized the polyclonal
antibody preparation 695. Selenium was measured using a modification of
the fluorometric assay of Koh and Benson (23, 24). The limit of
detection of this assay is 1 ng of selenium.
75Se Labeling of Mouse Plasma--
Adult mice of all
genotypes that were fed the chow diet were each injected
intraperitoneally with 15 µCi of [75Se]selenite (in
0.15 M NaCl). Blood was obtained from the mice 24 h
after 75Se administration. Plasma was separated by
centrifugation and subjected to SDS-PAGE. After being stained with
Coomassie Blue, the gel was dried and exposed to Kodak XAR film. In a
separate experiment, some of the 75Se-labeled plasma was
subjected to immunoprecipitation by polyclonal antibodies raised in
rabbits against rat selenoprotein P (25) and by polyclonal antibodies
raised in rabbits against human GSHPx-3 (pAb 3495, a generous gift of
K. R. Maddipati, Cayman Chemical, Ann Arbor, MI). The
immunoprecipitates were separated by SDS-PAGE, and
75Se-labeled proteins were identified by autoradiography.
Metabolic Fate of Selenium Administered by Gavage--
Male mice
of all three genotypes were fed the experimental diet supplemented with
0.1 mg of selenium/kg for 6-8 weeks from the time of weaning.
Then each was administered 15 µCi of [75Se]selenite (in
0.15 M NaCl) by gavage. They were housed individually for
24 h and were then anesthetized and exsanguinated by collection of
blood from the inferior vena cava. Liver, kidney, testis, and brain
were harvested and weighed. The 75Se content of plasma and
tissues was determined in a Commugamma 1282 (Amersham Biosciences).
Statistics--
Results were analyzed using Student's
t test or by analysis of variance with post hoc
analysis for statistical differences using the Scheffe test.
Significance was set at p < 0.05. Calculations were
done on a Macintosh G4 using Statview, version 5.0.1 (SAS Institute,
Cary, NC).
Generation of Sepp-deleted Mice--
Se-P knockout mice were
produced by homologous recombination using genomic DNA cloned from an
Sv-129 P1 library that had been mutated using the strategy shown in
Fig. 1. C57Bl/6 blastocysts were injected
with ES cells heterozygous for the Sepp mutation and then
implanted into pseudopregnant females. A male chimera was identified
among the offspring. This male was mated with two C57Bl/6j female mice,
and the heterozygote progeny (Sepp+/ Verification of Se-P Deletion--
Twenty-four hours after
75Se had been injected into animals of each genotype,
plasma was obtained and subjected to SDS-PAGE. Fig.
2A shows the
autoradiograph of the resulting gel. Both the Sepp+/+ and Sepp+/
Fig. 2D shows the results of measuring Se-P by competitive
radioimmunoassay of plasma samples from the three genotypes. A gene-dose effect is evident that mirrors the liver mRNA results (Fig. 2C). Thus, Se-P has been eliminated from the plasma of
the knockout mice, and its concentration is half that of the
wild types in the plasma of the heterozygotes.
Breeding of Se-P Knockout Mice--
Offspring of
Sepp+/ Biochemical Characterization of Selenium Status in Genetically
Altered Mice Fed Different Amounts of Selenium--
In the initial
experiments with our genetically altered mice (Tables I and II), we fed
mouse chow containing 0.29 mg of selenium/kg. The
Sepp
Sepp
Two selenoproteins contribute to plasma selenium: extracellular
glutathione peroxidase (GSHPx-3) and Se-P. Fig.
4A shows the selenium content
and glutathione peroxidase activity of plasma at different levels of
dietary selenium supplementation. The absence of Se-P from the plasma
of Sepp
Plasma glutathione peroxidase activity increased as the dietary
selenium supplement was increased from 0 to 0.1 mg/kg in wild type
(Sepp+/+) mice and maintained that level as
dietary selenium was increased further (Fig. 4A, right
panel). This result is consistent with the established
dietary requirement of 0.1 mg of selenium/kg in mice (26, 27).
Sepp+/
Liver selenium levels were not affected by deletion of Se-P, except
when dietary selenium was below the selenium requirement (Fig.
4B, left panel). The two
Sepp
Fig. 5 shows the selenium content of
testis, brain, kidney, and heart. Glutathione peroxidase activity was
also determined in these tissues, and its pattern (not shown) was
similar to that of the selenium concentrations. In each of these
tissues the values for Sepp+/+ and
Sepp+/ Tissue Uptake of 75Se--
To determine the effect of
Se-P deletion on the disposition of a single dose of selenium, a tracer
dose of 75Se as sodium selenite was administered by
gavage to mice being fed the diet supplemented with 0.1 mg of
selenium/kg. Tissues were harvested 24 h after administration, and
their 75Se content was determined; Fig.
6 shows the results. Liver showed a trend
of containing more of the 75Se administered in
Sepp Mice with Se-P deleted have been produced by homologous
recombination. These mice are viable but exhibit a profound alteration in selenium metabolism, which renders them intolerant of low dietary selenium intake. When intake is low they develop impaired movement and
coordination and do not maintain their weight. In addition, males have
sharply reduced fertility, even when fed enough selenium to prevent the
movement abnormality and weight loss.
These two phenotypes correlate with the effect of Se-P deletion on the
selenium content of the brain and testis (Fig. 5, B and A, respectively). Low selenium in the brain correlates
with the apparent neurological impairment, and low selenium in testis appears to underlie the impaired male fertility. Selenium is essential for spermatogenesis (28, 29). Brain and testis have been identified as
tissues that retain selenium well under conditions of extreme selenium
deficiency (30, 31). Therefore, a decade ago we compared the ability of
these two tissues to take up selenium administered in the form of
75Se-labeled Se-P (7). Both tissues took up the
75Se avidly. The brain was able to increase its uptake more
than 4-fold when selenium deficiency was imposed. In contrast, testis uptake, although brisk, was unaffected by selenium deficiency. These
results and the present ones suggest that the brain and testis remove
Se-P from plasma to acquire its selenium. Moreover, the brain would
appear to be able to up-regulate this uptake mechanism in selenium
deficiency. In addition, the neurological phenotype could be prevented
by feeding selenium to Sepp The severity of the effect of Se-P deletion on selenium concentration
forms a continuum in the tissues we examined. Although there were
severe effects on the testis and brain, the kidney was only moderately
affected and the heart was not significantly affected. This indicates
that other transport forms of selenium than Se-P exist and that each
tissue may have preferred forms of plasma selenium from which it
obtains the element.
In the mice studied here, plasma selenium was present as Se-P, GSHPx-3,
and small molecule forms. The origin of plasma Se-P is mostly liver (6)
and that of GSHPx-3 is kidney (32). The intestine is known to release a
small molecule form of selenium that is taken up by the liver (6), but
additional small molecule forms of selenium, other than excretory
metabolites, have not been detected. It can be inferred that the kidney
can also take up a small molecule form of selenium, because kidney is
the source of plasma GSHPx-3 and the selenium concentration of kidney
responds to dietary selenium in animals lacking Se-P (Fig.
5C).
There is no direct evidence that GSHPx-3 serves to supply tissues with
selenium. However, one finding in this study is consistent with such a
function. Plasma glutathione peroxidase activity did not rise to its
control level until 10 times the dietary requirement of selenium was
fed (Fig. 4A), whereas kidney selenium level rose to control
at only 2.5 times the requirement (Fig. 5C). If it is
assumed that synthesis and secretion of GSHPx-3 was normalized when
kidney selenium was at control levels, then increased removal of
GSHPx-3 from plasma must be invoked to explain the low glutathione peroxidase activity in plasma at that point. Mice with deletion of
GSHPx-3 will facilitate study of its putative transport role.
The liver has a central role in selenium metabolism. It receives small
molecule selenium directly from the intestine and is the major organ
for removal of selenium from the dietary form, selenomethionine,
via the trans-sulfuration pathway (33). The liver also is the principal
organ producing excretory metabolites of selenium to prevent the
accumulation of toxic levels of the element. Thus, the liver is the
portal through which selenium enters the body and the organ that
maintains its homeostasis through excretion of the element.
Secretion of Se-P into the plasma is another important
function of the liver. The increase of liver selenium concentration in
selenium-deficient mice with deletion of Se-P is therefore not
surprising (Fig. 4B). Selenium entering the liver in
Sepp The studies reported here address the selenium transport function of
Se-P. They indicate that the testis and brain have mechanisms for
acquiring selenium from plasma Se-P. The characterization of those
mechanisms will require additional research, as will assessment of the
putative oxidant defense role of Se-P.
/
) were generated. Two
phenotypes were observed: 1) Sepp
/
mice lost weight and developed poor motor coordination when fed diets
with selenium below 0.1 mg/kg, and 2) male
Sepp
/
mice had sharply reduced fertility.
Weanling male Sepp+/+,
Sepp+/
, and Sepp
/
mice were fed diets for 8 weeks containing <0.02-2 mg selenium/kg. Sepp+/+ and Sepp+/
mice had similar selenium concentrations in all tissues except plasma
where a gene-dose effect on Se-P was observed. Liver selenium was
unaffected by Se-P deletion except that it increased when dietary
selenium was below 0.1 mg/kg. Selenium in other tissues exhibited a
continuum of responses to Se-P deletion. Testis selenium was depressed
to 19% in mice fed an 0.1 mg selenium/kg diet and did not rise to
Sepp+/+ levels even with a dietary selenium of
2 mg/kg. Brain selenium was depressed to 43%, but feeding 2 mg
selenium/kg diet raised it to Sepp+/+ levels.
Kidney was depressed to 76% and reached
Sepp+/+ levels on an 0.25 mg selenium/kg diet.
Heart selenium was not affected. These results suggest that the
Sepp
/
phenotypes were caused by low
selenium in testis and brain. They strongly suggest that Se-P from
liver provides selenium to several tissues, especially testis and
brain. Further, they indicate that transport forms of selenium other
than Se-P exist because selenium levels of all tissues except testis
responded to increases of dietary selenium in
Sepp
/
mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice--
ES cell
transformation with SePCKNT and blastocyst injection were carried out
in the Mouse Core Facility at the University of Utah using published
procedures (18, 19). PCR assays were used to determine the genotype of
ES cells and adult mice. DNA was extracted from ES cells and from tail
biopsies using established procedures. DNA was resuspended in Tris-EDTA
buffer (10 mM Tris·Cl, 1 mM EDTA, pH
7.6). Approximately 1 µg of DNA was dissolved in 50 µl of PCR lysis
buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris·Cl (pH 8), 0.01% gelatin, 0.45% Nonidet P-40,
0.45% Tween 20), denatured at 95 °C for 5 min, and quick-chilled on
ice. Two µl of the denatured DNA solution was amplified for 35 cycles
(94 °C for 30 s, 66 °C for 20 s, 72 °C for 60 s) in a 10-µl reaction mixture. The primers used to screen for
ES-positive cells were MoSePS6 (5'-GAAGACTGTAATCGCTATAACCACTGTCCAG-3') and ACNeoS2 (5'-GGTGTTGGGTCGTTTGTTCGGATCG-3'). The primers used to
screen tissue DNA were MoSePS14 (5'-GCCATCAGGGCTCAGTGCAG-3'), MoSePA16
(5'-GTTCAAAGCCCAGGAATGCCACAG-3'), and ACNeoS2. PCR product sizes are:
wild type (Sepp+/+), 900 bp from MoSePS14 and
MoSePA16; homozygote (Sepp
/
),
500 bp from MoSePS14 and ACNeoS2; heterozygote
(Sepp+/
), 900 and 500 bp. An
additional PCR product of 2.1 kb is predicted for the primers MoSePS14
and MoSePA16 in Sepp+/
and
Sepp
/
mice. PCR conditions were not
optimized for production of this product.
and
Sepp
/
mice were transferred to the animal
facility at Vanderbilt University. The Vanderbilt University
Institutional Animal Care and Use Committee approved animal protocols
for studies at Vanderbilt, and the corresponding University of Utah
committee approved the protocols used to generate the knockout mouse.
The mice were housed in plastic cages with aspen shavings as bedding material. The light/dark cycle was 12 h:12 h. Mice received
pelleted rodent chow (selenium content, 0.29 ± 0.01 mg/kg) and
water ad libitum except when fed diets containing specific
amounts of selenium. Matings were set up between
Sepp
/
males and
Sepp
/
females, between
Sepp
/
males and
Sepp+/
females, between
Sepp+/
males and
Sepp
/
females, and between
Sepp+/
males and
Sepp+/
females. Pups were weaned 21 days after
birth and separated by sex.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) from
these matings were used to establish the Se-P knockout mouse
colony.
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Fig. 1.
Targeted disruption of the Sepp
gene. A, diagram of Sepp gene. Exons 2-5 are
designated by the gray rectangles, and selected restriction
sites are shown. The initiating ATG is 9 nucleotides 5' of the
AvrII site in exon 2. B, Neo® cassette
(represented by the cross-hatched rectangle) was inserted at
the AvrII site resulting in the insertion of a stop
codon in all three reading frames. C, the targeting vector
was made by ligation of the Neo-containing Sepp gene with
TK-2 and pBluescript (represented by diagonally striped
rectangles) as described under "Experimental
Procedures."
plasma samples yielded bands characteristic for Se-P and GSHPx-3. Sepp
/
plasma exhibited only the band of
75Se radioactivity corresponding to glutathione peroxidase.
The Se-P band was not present in the Sepp
/
lane. The identity of each of the radioactive proteins was verified by
precipitation using specific antibody preparations (Fig.
2B). Northern analysis of liver RNA was carried out and
showed a gene-dose effect with no signal evident in the
Sepp
/
lane (Fig. 2C).
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Fig. 2.
Effect of gene deletion (Sepp) on
Se-P. A, autoradiograph of gel after SDS-PAGE of plasma
collected from mice that had been injected 24 h earlier with
[75Se]selenite. Arrows indicate migration of
marker proteins, and protein mass is indicated in kDa. The
increase in 75Se in the band at the GSHPx position in
Sepp /
mice was probably caused by diversion
of 75Se from Se-P to GSHPx-3. B, autoradiographs
of gels after SDS-PAGE of immunoprecipitated pellets of Se-P and
GSHPx-3 from plasma labeled as in A. C, Northern
analysis of liver RNA. D, radioimmunoassay of plasma. One
unit represents the amount of Se-P in 1 ml of a reference mouse plasma
(Harlan, Indianapolis, IN). Values are means ± S.D. The groups
are significantly different from each other by the Student's
t test, p < 0.05.
and Sepp
/
animals fed rodent chow were characterized. The ratio of male to female
progeny was ~50/50 regardless of the genotype of the parents (data
not shown). Mating of Sepp+/
males and
Sepp+/
females resulted in viable pups with
the predicted genotype distribution (Table
I, top). Those pups survived to weaning
(Table 1, bottom). Mating of Sepp+/
males and Sepp
/
females produced fewer
Sepp
/
pups than predicted, and 31% of these
homozygous pups died before weaning. When
Sepp
/
males were mated with
Sepp+/
and Sepp
/
females over a period of 6 months, only one litter was born from 18 mating pairs (Table II). In contrast,
Sepp+/
males produced many pregnancies with
Sepp+/
and Sepp
/
females. These results indicate that female homozygotes have difficulties producing and raising homozygote pups and that male homozygotes have sharply reduced fertility.
Genotype distribution and viability of pups produced by
Sepp+/ sires
Fertility of Sepp/
and Sepp+/
mice
/
mice grew as well as the
Sepp+/+ mice (Fig.
3A) and appeared healthy.
However, when we began feeding a selenium-deficient diet containing
<0.02 mg of selenium/kg, the Sepp
/
mice did
not survive for more than a few weeks, whereas
Sepp+/
and Sepp+/+ mice
survived. To investigate this sensitivity of
Sepp
/
mice to selenium deficiency, selenium
and glutathione peroxidase were measured in tissues from male mice of
all three genotypes that for 8 weeks had been fed diets containing
amounts of selenium ranging from deficient to 20 times the dietary
requirement. These mice were obtained by mating
Sepp+/
males with
Sepp+/
females.
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Fig. 3.
Weights of Sepp /
(squares) and Sepp+/+
(circles) mice fed different diets and different
amounts of selenium for the 8 weeks after weaning. Values are
means ± S.D., n = 4-6 at weaning. A
cross indicates death of an animal. A, male
(filled symbols) and female (open symbols)
animals fed chow diet containing 0.29 mg of selenium/kg. B,
male mice fed a selenium-deficient diet. C, male mice fed a
diet with 0.05 mg of selenium/kg. D, male mice fed a diet
with 0.10 mg of selenium/kg.
/
mice fed the selenium-deficient diet
(no selenium supplementation) gained weight for about a week and then
began to suffer from loss of motor coordination and weight (Fig.
3B). When mice had lost 20% of their highest recorded body
weight, they were euthanized. Their average survival time from weaning
was 15 days. Sepp
/
mice fed the diet with
0.05 mg of selenium added/kg also had decreased survival times; 2 of 4 mice were sacrificed because of 20% weight loss, one at 3 weeks and
the other at 6.5 weeks (Fig. 3C). Thus only 2 Sepp
/
mice fed this diet remained alive at
the 8-week time point. Animals fed an 0.1 mg of selenium or more/kg
diet all survived to 8 weeks without loss of coordination, and weights
were similar for all genotypes (Fig. 3D).
/
mice is reflected in the very low
plasma selenium concentrations measured at all levels of selenium
supplementation (Fig. 4A, left panel). Selenium
concentrations in Sepp+/
mouse plasma were
intermediate between the homozygote and wild type concentrations at
each supplementation level. This finding is consistent with the
gene-dose effect indicated in Fig. 2D by the Se-P
concentrations in plasma.
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Fig. 4.
Selenium concentration and glutathione
peroxidase activity in plasma (A) and liver (B)
of mice with deletion of Se-P (Sepp+/+,
circles; Sepp+/ ,
diamonds; Sepp
/
,
squares). Mice were fed, for the 8 weeks following
weaning, on experimental diets with selenium added as indicated in the
figure. Values are means ± S.D., n = 4-6 except
value marked with an asterisk where n = 2. The other two mice died at 3 and 6.5 weeks. All values shown in
A, left panel, at each dietary selenium level are different
by the Scheffe test (p < 0.05), except
Sepp+/+ and Sepp+/
at 0 and 0.05 mg of selenium/kg. In A, right panel,
Sepp
/
values at 0.05, 0.10, 0.25, and 0.50 mg of selenium/kg are different by the Scheffe test (p < 0.05) from the corresponding Sepp+/+ values,
but no difference between them is present at 1.0 or 2.0 mg of
selenium/kg. The liver selenium concentrations of the mice that died at
3 and 6.5 weeks were 910 and 1000 ng/g, respectively. In B, left
panel, the Sepp
/
values at 0.05 and
0.25 mg of selenium/kg are different from the corresponding
Sepp+/+ and Sepp+/
values by the Scheffe test (p < 0.05). In B,
right panel, the Sepp
/
value at 0.05 mg
of selenium/kg is different from the corresponding
Sepp+/+ and Sepp+/
values by the Scheffe test (p < 0.05). Other values at
each dietary selenium level are not significantly different from each
other.
plasma glutathione peroxidase
activities were not significantly different from activities in the
Sepp+/+ mice. Sepp
/
mice had lower plasma glutathione peroxidase activities than Sepp+/+ mice at levels of selenium
supplementation below 1.0 mg/kg. These results indicate that Se-P is
needed to achieve normal plasma activity of GSHPx-3 at dietary selenium
concentrations up to 5 times the requirement but not when the dietary
selenium concentration is 10 times the required concentration.
/
animals in the 0.05 mg selenium/kg
diet group that survived for 8 weeks had levels that were greater than
the averages of the other groups. The liver selenium levels of the two
Sepp
/
animals that did not survive 8 weeks
were also greater than the 8-week averages of the other animals (see
legend for Fig. 4). Liver glutathione peroxidase activities had the
same pattern (Fig. 4B, right panel) as liver
selenium concentrations. These results indicate that the
Sepp
/
liver retains more selenium than the
Sepp+/+ liver during selenium deficiency and
that deletion of Se-P does not cause a decrease in liver selenium
content at any dietary selenium level. They also demonstrate that
deletion of Se-P does not impair regulation of selenium concentration
in liver when higher levels of selenium are fed.
mice were similar. The selenium
difference between the Sepp+/+ or
Sepp+/
mice, on the one hand, and the
Sepp
/
mice, on the other hand, varied by
tissue. It was greatest in testis, with selenium concentration being
19% of Sepp+/+ when selenium was fed at the
dietary requirement. Moreover, increasing dietary selenium 20-fold did
not raise the level of testis selenium into the
Sepp+/+ range. The brain was also severely
affected with its selenium concentration being 43% of control at the
dietary requirement. However, brain selenium was raised into the
Sepp+/+ range by increasing dietary selenium
20-fold. Kidney selenium was less affected, being 76% of
Sepp+/+ when the dietary selenium requirement
was fed. Moreover, increasing dietary selenium to just 2.5-fold the
requirement raised the selenium content of
Sepp
/
kidney to the same level found in
Sepp+/+ kidney. Heart selenium concentration was
not significantly affected by genotype. These results indicate that
Se-P facilitates selenium accumulation in testis, brain, and kidney.
Heart selenium content does not appear to depend on Se-P. Thus, the
tissues studied vary in their dependence on Se-P to maintain their
selenium concentrations.
View larger version (22K):
[in a new window]
Fig. 5.
Selenium concentrations in the testis
(A), brain (B), kidney (C), and
heart (D) of mice with deletion of Se-P. The results
are for the same groups depicted in Fig. 4. Two of the
Sepp /
mice fed the diet with 0.05 mg of
selenium added/kg survived 8 weeks; the average of their values is
indicated by the symbol with an asterisk. Testes
were not available for analysis for all animals. Values are means ± S.D., n = 4-6. Some S.D. are too small to be
displayed on the figure. In A, all
Sepp
/
values are different from
corresponding Sepp+/+ and
Sepp+/
values by the Scheffe test,
p < 0.05. In B,
Sepp
/
values from 0.05 to 1.0 mg of
selenium/kg are different from corresponding
Sepp+/+ and Sepp+/
values by the Scheffe test, p < 0.05. The
Sepp
/
value at 2.0 mg of selenium/kg is not
significantly different from the Sepp+/+ value.
In C, the Sepp
/
values at 0.05, 0.10, and 0.5 are different from corresponding
Sepp+/+ and Sepp+/
values by the Scheffe test, p < 0.05. In D,
none of the values at individual dietary levels are significantly
different from one another.
/
mice than it did in
Sepp+/
or Sepp+/+ mice.
Brain and testis contained less 75Se in
Sepp
/
mice than in the other two genotypes.
These results show that absorbed selenium is readily taken up by the
liver in Sepp
/
mice but is less well taken
up and/or retained by the brain and testis. These results are
consistent with the tissue selenium levels shown in Figs. 4 and 5.
View larger version (25K):
[in a new window]
Fig. 6.
75Se in mouse tissues
24 h after administration of a submicrogram dose of
75Se-labeled selenite by gavage. Animals
had been fed the experimental diet containing 0.1 mg of selenium
added/kg for 6-8 weeks after weaning. Values are means ± S.D.,
n = 4 for the total organ (or pairs of organs).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice at the
level of dietary requirement or higher. This suggests that the brain
can take up other plasma forms of selenium than Se-P and therefore
utilizes two or more mechanisms to obtain the selenium it needs. No
evidence was found that the testis is able to utilize a form of
selenium other than Se-P.
/
mice is presumably diverted from
export as Se-P to hepatic selenoproteins such as glutathione
peroxidase. Se-P deletion did not interfere with the regulation of
liver selenium content when levels above the dietary requirement for
selenium were fed. Whether the liver selenium concentration was
maintained by release of selenium in a form that could be taken up by
other tissues or by production of excretory metabolites was not
addressed in these experiments. Determination of this will be important
in the elucidation of selenium homeostasis.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Kirk R. Thomas for help in generating the mutant mice as well as for very helpful discussions and to Dr. Marla J. Berry for suggesting the collaboration that led to this study.
![]() |
Addendum |
---|
During final preparation of this manuscript, a report on Se-P gene deletion in mice was published by Schomburg et al. (34).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 ES02497 and P30 ES00267. It was presented at Experimental Biology 2002, the annual meeting of the Federation of American Societies for Experimental Biology, on April 22, 2002 and published in abstract form (Hill, K. E., Zhou, J., McMahan, W. J., Motley, A. K., Atkins, J. F., Gesteland, R. F., and Burk, R. F. (2002) FASEB J. 16, A605 (abstr.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by National Institutes of Health Grant GM48152.
Supported by National Institutes of Health Grant GM61200.
** To whom correspondence should be addressed: Medical Center North, C-2104, Vanderbilt Medical Center, Nashville, TN 37232-2279. Tel.: 615-343-7740; Fax: 615-343-6229; E-mail: raymond.burk@vanderbilt.edu.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M300755200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Se-P, selenoprotein P; Sepp, gene encoding selenoprotein P; GSHPx, glutathione peroxidase; ES, embryonic stem.
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