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Reproductive Biology Section, Departments of Obstetrics/Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8063
Address all correspondence and requests for reprints to: Pinar H. Kodaman, Reproductive Biology Section, Department of Obstetrics and Gynecology, Yale University School of Medicine, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: kodamaph{at}biomed.med.yale.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sequential two electron oxidation of ascorbic acid during radical scavenging produces dehydroascorbic acid (DHAA). Although an unstable compound under physiological conditions (t1/2 = 6 min), DHAA is the major transportable form of ascorbate for certain cell types, including erythrocytes (7), neutrophils (8, 9), and endothelial cells (10). Intracellular DHAA is reduced to ascorbate by a glutathione-and NADH-dependent semidehydroreductase system (11, 12) that allows for the recycling of ascorbic acid while simultaneously clearing plasma of DHAA, which at high concentrations, is cytotoxic (13, 14). In the absence of reduction to ascorbic acid, DHAA is irreversibly degraded to 2,3-diketogulonic acid.
The uptake of ascorbate in either reduced or oxidized form is requisite for maintaining adequate intracellular antioxidant tone. While species such as rodents produce ascorbic acid via hepatic synthesis (15), primates, guinea pigs, and fruit-eating bats must acquire this vitamin through dietary intake as they lack L-gulonolactone oxidase, the terminal enzyme in the L-ascorbic acid biosynthetic pathway. Regardless of its route of acquisition, ascorbic acid is accumulated in ovarian cells by membrane transporters against a large concentration gradient even in animals in which ascorbate is not a dietary requirement as synthesis occurs only in the liver (16). The ascorbic acid transporter has not been cloned, whereas GLUT1 has been implicated as the putative DHAA transporter in certain cell types, including neutrophils (8, 17, 18). Osteoclasts (19) and luteal cells (20) also appear to accumulate DHAA via facilitative hexose transporters.
Our recent studies demonstrated that granulosa cells transport ascorbic acid in an endocrine-regulated manner (21); however, to our knowledge, neither the follicle nor granulosa cells have been examined for their ability to transport DHAA. The objective of the present studies was to examine DHAA transport in granulosa cells as a potential mechanism for the accumulation and conservation of cellular ascorbate. Furthermore, it was of particular interest to determine whether DHAA transport in granulosa cells is a constitutive or hormone-regulated process and whether DHAA uptake occurs via facilitative hexose transporters in granulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P ]deoxy (d)-CTP (3000 Ci/mmol) and
[
-32P]-ATP (3000 Ci/mmol) were also purchased from
Amersham Pharmacia Biotech, and all other chemical
reagents were obtained from Sigma Chemical Co. (St. Louis,
MO). A plasmid containing subcloned human Glut1 complementary DNA (1.8 kb) was a gift from Graeme Bell (University of Chicago, Chicago, IL), while polyclonal rabbit antibodies directed against GLUT1 and GLUT4 were acquired from Dena Yver (NIH, Bethesda, MD).
Animals and preparation of granulosa cells
Immature (21- to 23-day-old) female rats (SD strain,
Taconic Farms, Inc., Germantown, NY) were pretreated for 4
days with diethylstilbestrol (DES) delivered by 1 cm SILASTIC brand
capsules (Dow Corning Corp., Midland, MI) implanted under
the skin of the upper back (22) after anesthesia with methoxyflurane
(Malinckrodt Veterinary, Mundelein, IL). Animals were housed and cared
for in the fully accredited facilities operated by the Animal Resource
Center (Yale University School of Medicine, New Haven, CT). All
treatments and procedures were in accordance with the NIH guide for the
Care and Use of Laboratory Animals and a protocol approved by the Yale
University Animal Care Committee.
Before they were killed, animals were anesthetized with a solution of ketamine hydrochloride (Quad Pharmaceuticals, Indianapolis, IN), xylazine hydrochloride (Rompun; Miles, Inc., West Haven, CT), and 0.9% NaCl (0.5:0.11:1.39, vol/vol/vol; 0.25 ml/animal) and perfused with saline to remove circulating blood cells, as erythrocytes and leukocytes are known to transport substantial amounts of DHAA (17, 23). The ovaries were removed, placed in ice-cold DMEM-F12 culture medium (Life Technologies, Inc., Grand Island, NY) supplemented with 0.1% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin. Ovaries were trimmed of their bursae and surrounding fat tissue, and follicles were punctured with a 27-gauge needle to release granulosa cells, which were harvested as previously described (21, 24, 25). In brief, released cells were kept on ice, transferred into a sterile hood, filtered through a 70 µm Falcon nylon cell strainer (Becton Dickson Labware, Lincoln Park, NJ), and washed twice with sterile culture medium before overnight culture.
Cell culture protocol
Granulosa cells were plated (1–4 x
106/ml/well) in 24-well tissue culture dishes and incubated
overnight at 37 C under a humidified atmosphere of 95% air/5%
CO2 in the culture medium described above. Insulin (10
nM), IGF-I (30 ng/ml), IGF-II (30 ng/ml) and/or FSH (50
ng/ml) were added to certain wells for overnight incubation.
Analysis of viability, proliferation, and apoptosis
Cell viability was determined by exclusion of Trypan blue (26)
and by the ability of cells to metabolize
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) (27, 28). Mitochondrial metabolism of MTT results in the
formation of a tetrazolium precipitate that is dissolved with
isopropanol and quantified by colorimetry (27, 28). The MTT assay was
also used to assess treatment-induced proliferation.
After 24 h of culture in the presence and absence of various treatments indicated in the text, cells were also assessed for apoptosis by fixation in 3% paraformaldehyde, permeabilization with 1% Triton X-100 in PBS/10 mM glycine, and staining and mounting with Vectashield Mounting Medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA) (29). Nuclei in replicated experiments were examined independently by two separate investigators using fluorescence microscopy. Cells were also assessed for apoptosis by flow cytometry following fixation in ethanol and staining with propidium iodide as previously described (30).
Dehydroascorbic acid uptake
Before measurement of DHAA uptake, wells were washed once with
HBSS, pH 7.4, supplemented with 0.5 mM thiourea and 0.1%
BSA. Thiourea was used to stabilize ascorbic acid before the addition
of ascorbate oxidase, which catalyzes the conversion of ascorbic acid
to DHAA. All uptake experiments were carried out in this medium, which
was additionally supplemented with ascorbate oxidase (2.0 U/ml), and in
some cases, D-glucose (2.5–10 mM) and insulin,
IGF-I, IGF-II, or FSH at the concentrations indicated above.
Dehydroascorbic acid uptake was initiated by the addition of
[14C]-ascorbic acid (0.15 µCi; specific activity:
13.7–18.2 mCi/mmol). Our previous studies demonstrated the rapid
conversion (45 sec) of 300 µM ascorbic acid to DHAA by
ascorbate oxidase (2.0 U/ml) (20), and thus, preprepared DHAA was not
used because of its lack of purity in solution, which results from the
compound’s inherent instability. In the presence of ascorbate oxidase,
there is essentially no ascorbic acid available for uptake as shown by
HPLC of media within 1 min of incubation, which revealed that 90.7% of
the radioactivity was DHAA and only 2.6% was in the form of ascorbic
acid (20).
Total DHAA concentration ranged from 30 to 3000 µM for the kinetics experiments and was 300 µM for all other experiments. DHAA concentration was adjusted by the addition of unlabeled ascorbate. In certain experiments, uptake was assessed in the presence and absence of the glucose transport inhibitors phloretin (200 µM) and cytochalasin B (10 nM), or the Na+/K+ ATPase inhibitor ouabain (100 µM). Following labeling for 0–60 min, cells were lysed with 1 N NaOH, and intracellular [14C]-DHAA was quantified as previously described (20). Nonspecific background radioactivity was assessed in identically treated but nonincubated cells.
HPLC analysis
Following 2 h of labeling, cells were extracted on ice for
30 min with 200 µl extraction buffer (1.5% metaphosphoric acid, pH
3.5, 0.3 mM ascorbic acid, 0.1 mM EDTA, 0.5
mM thiourea). The extract was subsequently combined with
667 µl acetonitrile, centrifuged at 10,000 x g for
10 min (4 C), and the resulting supernatant was filtered twice through
a 0.2 µm filter. The filtrate (500 µl) was immediately analyzed by
HPLC. Dehydroascorbic acid and its metabolites were separated on a
Phase Sep S5 NH2 column (Waters Millipore Corp., Milford,
MA) with acetonitrile: 0.05 M
KH2PO4 (70:30; w/w) as the mobile phase and at
a flow rate of 1.5 ml/min, as previously described (31). Fractions were
collected at 1-min intervals for 20 min, and ascorbic acid was detected
at 268 nm, whereas DHAA and diketogulonic acid were identified based on
retention times relative to ascorbic acid (31). The external standard
was [14C]-ascorbic acid (10,000 cpm; 300
µM) in HBSS diluted with extraction buffer and
acetonitrile, as above.
RNA extraction and Northern blotting
Total RNA was extracted from granulosa cells with Trizol reagent
(Life Technologies, Grand Island, NY). All RNA samples had
a 260/280 ratio greater than 1.8. For Glut1 analysis, equal amounts of
total RNA (10 µg) from each sample were electrophoresed in a 1%
agarose gel and capillary transferred overnight onto Zetabind membrane
(Cuno, Meriden, CT). The membranes were subsequently prehybridized for
4 h at 60 C in 0.5 M sodium phosphate, pH 6.8, 7%
SDS, 1% BSA, and 1 mM EDTA before hybridization overnight
with a 1.8 kb Glut1 complementary DNA probe (2 x 106
cpm/ml) labeled with [-32P]dCTP by random
oligonucleotide priming using a kit obtained from Roche Molecular Biochemicals (Indianapolis, IN). The blots were subsequently
washed 3 times 30 min in 40 mM sodium phosphate, pH 6.8,
5% SDS, 0.5% BSA, and 1 mM EDTA at 60 C, followed by 3
times 30 min in 40 mM sodium phosphate, pH 6.8, 1% SDS, 1
mM EDTA at 60 C.
For all Northern analyses, loading was controlled for by hybridization to 28S ribosomal RNA after stripping the blots with 0.1 x SSC-0.5% SDS (3 times 30 min; 80 C). Blots were quantified by densitometry after multiple exposures to ensure quantitation in the linear response range.
Membrane protein extraction and Western blotting
Cultured granulosa cells exposed to the various treatments
described in the text were frozen (-20 C) in buffer containing 20
mM Tris-HCl, 1 mM EDTA, 225 mM
sucrose, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, and 25 µg/ml pepstatin, before
homogenization with a glass-Teflon homogenizer. Membrane proteins were
extracted as previously described (32), and protein concentration was
assessed using the Bio-Rad reagent (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of protein (25 µg/sample)
were fractionated by 10% SDS-PAGE under reducing conditions and
transferred onto Nytran membrane (Schleicher & Schuell, Inc., Keene, NH). For both GLUT1 and GLUT4 Western analyses,
blots were blocked for 2 h in blocking solution (Schleicher & Schuell, Inc.) before incubation with primary antibody
(1:1000) for 1 h at room temperature. Detection was achieved using
horseradish peroxidase-conjugated goat-antirabbit antibody and
Starlight substrate with Background Minimizer (Schleicher & Schuell, Inc.). The blots were exposed multiple times to ensure
that quantitation was within the linear response range, and visualized
bands were then quantified by densitometry. Prestained SDS-PAGE
standards (Bio-Rad Laboratories, Inc.) were used to
confirm the molecular weights of the detected bands, which were
approximately 45 kD for GLUT1 and GLUT4 and in concordance with those
previously reported (33).
Statistical analysis
Granulosa cells from several rats were pooled, and equal
aliquots were exposed to each treatment in triplicate. Each experiment
was repeated at least three times. Statistical significance between
treatments within an experiment was determined by one-way repeated
measures ANOVA. Post hoc comparisons of multiple treatments were made
by the Bonferroni t test method. Treatment differences
between experiments were determined by one-way ANOVA, and post hoc
comparisons were made with Dunnett’s method. In all analyses,
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). All subsequent uptake experiments
were conducted with 1–2 x 106 cells and for 30 min
of incubation, unless otherwise indicated. Kinetics studies
demonstrated that transport of DHAA was a substrate-dependent and
saturable process with an estimated Michaelis constant (Km)
of 500 µM and a maximum velocity of 19
pmol/106/min. Due to the inherent instability of DHAA,
these kinetics values are broad estimates; however, the fact that
uptake was linear for 30 min and cell number-dependent serves as the
basis for the semiquantitative analysis of DHAA transport. HPLC
analysis of cellular contents following a 2 h period of
[14C]-labeled DHAA uptake revealed that 89.4 ±
0.5% of intracellular radioactivity was in the form of ascorbic acid,
while 4.5 ± 0.1% was DHAA.
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shows that 24 h
preincubation of granulosa cells in the presence of FSH (50 ng/ml) and
IGF-I (30 ng/ml) together stimulated DHAA uptake (158 ± 16
pmol/106 cells) compared with the control (43 ± 8
pmol/106 cells). Each agent alone produced small, but not
significant effects on DHAA transport (60 ± 12 and 54
pmol/106 cells, respectively). Both insulin (10
nM) and IGF-II (30 ng/ml) were without significant effect
alone and in combination with FSH as was LH (1 µg/ml) (data not
shown).
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Effects of glucose and inhibitors of glucose transport on DHAA
uptake
Figure 3 illustrates that following
24 h preincubation with FSH and IGF-I, uptake of DHAA was
significantly reduced in the presence of the glucose transport
inhibitors phloretin and cytochalasin B (19.2 ± 2.6 and 12.6
± 4.0 pmoles/106 cells, respectively) compared with the
control (153 ± 17 pmol/106 cells). DHAA uptake was
not significantly affected by preincubation with ouabain, an inhibitor
of Na+/K+ ATPase, and no significant
cytotoxicity was observed following incubation with ouabain, phloretin,
or cytochalasin B as assessed by the MTT assay (data not shown). The
relationship between facilitative hexose transporters and DHAA uptake
in granulosa cells was further examined by assessing DHAA transport in
the presence of increasing concentrations of D-glucose. As
shown in Fig. 4, DHAA uptake was
significantly inhibited by glucose in a dose-dependent manner with an
IC50 of approximately 2.5 mM.
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).
Western analysis of membrane proteins extracted from granulosa cells
revealed that GLUT1 protein levels were also significantly increased
(1.31 ± 0.04 fold of control) following incubation with FSH and
IGF-I together as illustrated in Fig. 6.
IGF-I alone also had a small, but significant effect on protein levels
of GLUT1 (Fig. 6). GLUT4 was undetectable in the same granulosa cells
by Western analysis (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of granulosa cell extracts following 2 h of DHAA uptake revealed that essentially all (ca 90%) of the transported DHAA was reduced to ascorbic acid. The intracellular reduction of DHAA to ascorbic acid allows for the conservation of ascorbate, and the reduced form of ascorbic acid is preferentially retained within oocytes (18). It is well known that other cell types, including neutrophils (9) and luteal cells (20), also accumulate ascorbate by the transport of DHAA. Recycling of DHAA may also serve to circumvent the potentially toxic effects of high local concentrations of DHAA or its breakdown products, such as 2,3-diketogulonic acid (35).
Dehydroascorbic acid uptake by granulosa cells appears to occur in an endocrine-regulated manner as FSH and IGF-I significantly stimulated increased transport. The cooperative effect of FSH and IGF-I on DHAA transport is similar to that seen with ascorbic acid transport by rat granulosa cells (21) and also agrees with the cooperative effects of FSH and IGF-I on granulosa cell differentiation (36, 37, 38, 39) and steroidogenesis (40) and on the inhibition of granulosa cell apoptosis (41) Recent studies have demonstrated that FSH responsiveness is essential for the prevention of follicular atresia and for selection of the dominant follicle (42), and it appears that this effect may be mediated by IGF-I (41, 43), which is produced locally by granulosa cells (44, 45). Tilly et al. (6) have suggested that the mechanism by which FSH prevents follicular atresia may be through the inhibition of granulosa cell apoptosis, and interestingly, ascorbic acid and other inhibitors of oxidative stress were able to mimic the FSH-induced blockade of apoptosis in granulosa cells of cultured follicles (6). These data, in conjunction with the findings of the present study, further underscore the importance of endocrine-regulated ascorbate transport and maintenance of adequate intracellular antioxidant tone for successful follicular development.
The present studies demonstrate that DHAA uptake in granulosa cells occurs via hexose transporters as DHAA transport was dose dependently inhibited by D-glucose and inhibitors of glucose transport. Uptake was not significantly affected by inhibition of Na+/K+ ATPase by ouabain, indicating that the glucose transporter used for DHAA uptake is not sodium-coupled unlike that for ascorbic acid uptake (21). Facilitative glucose transporters characterized to date include GLUTs 1 through 5 and GLUT7 (46). GLUT1 was chosen for further study, as it is widely expressed and has been implicated as a possible candidate for the DHAA transporter in neutrophils (8, 17, 18). Furthermore, Vera et al. (18) have shown that Xenopus laevis oocytes expressing Glut-1 were able to accumulate ascorbate by transport of DHAA.
It appears that GLUT1 may mediate DHAA transport in granulosa cells, as Northern analysis revealed cooperative induction of Glut1 expression by FSH and IGF-I that mirrored the cooperative increase in DHAA uptake stimulated by these hormones. Western analysis showed that protein levels of GLUT1 were similarly increased following treatment with both FSH and IGF-I. While GLUT4 has also been implicated as a DHAA transporter (18), we were unable to detect GLUT4 in rat granulosa cells by Western analysis; however, the possibility remains that other GLUT transporters may also mediate DHAA transport in granulosa cells.
The uptake of DHAA via glucose transporters has implications for ascorbate accumulation under hyperglycemic conditions. Nearly one-third of women with insulin-dependent diabetes mellitus have some form of menstrual dysfunction (47, 48, 49), and there is evidence that plasma levels of ascorbic acid are decreased (50, 51), while those of DHAA are abnormally increased (52, 53) in certain diabetic patients. The elevation of circulating DHAA may be secondary to both increased oxidation of ascorbic acid and elevated glucose levels, which competitively inhibit the cellular uptake of DHAA, and thus may further compromise antioxidant tone and contribute to the oxidative stress-related complications of diabetes.
In summary, the present studies demonstrate that rat granulosa cells accumulate ascorbic acid via the transport of DHAA through an insulin-insensitive, facilitative glucose transporter. GLUT1 is a candidate for the DHAA transporter in rat granulosa cells, where it is regulated by the cooperative actions of FSH and IGF-1 as is the uptake of DHAA. The ability of granulosa cells to transport DHAA in an endocrine-regulated manner may be invaluable in the maintenance of adequate antioxidant tone within the developing follicle.
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Acknowledgments |
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Footnotes |
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Received September 16, 1998.
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