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Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Dr. K. M. J. Menon, 6428 Med Sci I, 1300 Catherine Street, University of Michigan, Ann Arbor, Michigan 48109. E-mail: kmjmenon{at}umich.edu
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Abstract |
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Introduction |
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It is now well established that the steroidogenic activity of the theca-interstitial cells is regulated by LH and the insulin/insulin-like growth factor I (IGF-I) system (4, 5, 6, 7, 8, 9, 10). Elevated insulin levels secondary to metabolic resistance to insulin have been shown to be associated with conditions that produce hyperandrogenic states (11), suggesting that the stimulatory effects of insulin on androgen production by theca-interstitial cells might lead to anovulation. Another regulator of steroidogenic activity of most steroid-producing cells is plasma lipoproteins, low density lipoprotein (LDL), and high density lipoprotein (HDL). Because cholesterol is an essential precursor of steroid hormones in all steroidogenic tissues, the uptake of cholesterol from plasma plays a crucial role in steroid hormone biosynthesis. Our previous studies have shown that the rat ovarian cells acquire cholesterol from both LDL and HDL (12, 13, 14, 15, 16). In the ovary, the delivery of cholesterol from LDL uses the endocytotic internalization pathway similar to that described by Brown and Goldstein for cultured fibroblasts (17). The mechanism of uptake from HDL involves a specialized pathway, requiring interaction with a cell surface receptor (16, 18). The HDL receptor (SR-BI) messenger RNA (mRNA) was shown to localize in theca-interstitial cells as well as in luteinized granulosa cells, cell types known to use cholesterol for steroid hormone production (19). Furthermore, the expression of HDL receptor mRNA was shown to be induced by hCG (18, 19, 20). Addition of HDL to cultured theca-interstitial cells increases androgen output (21). Thus, cholesterol-carrying lipoprotein is a physiological regulator of theca-interstitial cell androgen synthesis.
Under pathological conditions, androgen production by theca-interstitial cells exceeds physiological levels, which, in turn, disturbs ovarian functions, including ovulation (3). As high circulating levels of insulin have been shown to be associated with increased synthesis of androgens by the theca-interstitial cells, the present studies were undertaken to examine the role of insulin in cholesterol transport into theca-interstitial cells. Specifically, we examined the effect of insulin and hCG on the expression of HDL receptor mRNA and intracellular cholesterol accumulation. Our results show that insulin produces an increase in the expression of HDL receptor mRNA in theca-interstitial cells in vitro and in vivo. Furthermore, insulin treatment increases the intracellular accumulation of cholesterol in theca-interstitial cells. The present study also shows that insulin potentiates hCG- mediated induction of HDL receptor mRNA in theca-interstitial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
[4-14C]Cholesterol (55 mCi/mmol) and
[1
,2-N-3H]cholesteryl loleate (48
Ci/mmol) were purchased from Amersham Pharmacia Biotech
(Elk Grove, IL). Peroxidase and cholesterol oxidase were obtained from
Calbiochem (La Jolla, CA). Cholesterol esterase, bovine
insulin, and hCG were purchased from Sigma (St. Louis,
MO). [-32P]Deoxy-CTP (3000 Ci/mmol) was
obtained from ICN Biomedicals, Inc. (Irvine, CA). The RTS
RadPrime DNA labeling kit, medium 199, L-glutamine,
McCoy’s 5A medium, and TRIzol reagent were obtained from Life Technologies, Inc. (Gaithersburg, MD). OCT compound was a
product of Miles (Elkhart, IN). X-Omat AR film and NTB-2 emulsion were
purchased from Eastman Kodak Co. (Rochester, NY).
Ribonuclease A, ribonuclease T1, deoxyribonuclease I, and proteinase K
were obtained from Roche (Indianapolis, IN). Collagenase
(CLS I; 260 U/mg) was purchased from Worthington Biochemical Corp. (Freehold, NJ). All other chemicals were obtained from
Fisher Scientific (Pittsburgh, PA) or
Sigma.
Cholesterol determination
Twenty-five-day-old rats, each group consisting of six rats,
were treated with insulin and/or hCG and killed at 0, 4, 8, and 12
h after treatment. When whole ovaries were used, the follicles were
punctured, follicular fluid was removed, and the tissue was
cut into 0.5-mm pieces with a razor blade and rinsed thoroughly to
remove any associated granulosa cells. Minced tissue was homogenized
with 10 vol homogenization buffer (0.3 M sucrose,
25 mM Tris-HC1, and 1 mM
EDTA, pH 7.4). Lipid extraction was performed using the method of Bligh
and Dyer (22), with the addition of 20,000 cpm
[3H]cholesterol ester and 20,000 cpm
[14C]cholesterol to monitor recovery. After
extraction with chloroform/methanol, the chloroform layer was collected
and evaporated at 45 C under a gentle stream of nitrogen. The pellet
was dissolved in 400 µl isopropanol and assayed for total and free
cholesterol by the enzymatic assay described by Deacon and Dawson
(23). Briefly, 1 ml working reagent (6
mM sodium cholate, 1.5% Triton X-100, 7.5
mM phenol, 0.5 mM
4-aminophenazone, 16.4 U horseradish peroxidase, and 0.08 U cholesterol
oxidase) was added to 0.18 ml of each standard or sample, and the
absorbance was measured at 500 nm. For total cholesterol determination,
the samples were hydrolyzed with cholesterol esterase to convert
esterified cholesterol to free cholesterol, and the resulting total
cholesterol was assayed by the enzymatic method described above.
Protein determination
Protein was assayed by the bicinchoninic acid method
(Pierce Chemical Co., Rockford, IL).
Northern blot analysis of SR-BI
Total RNA was extracted from ovaries pooled from six rats in
each treatment group, using the procedure of Chomczynski and Sacchi
(24). The SR-BI complementary DNA (cDNA) probe (308 bp)
was radiolabeled using [-32P]deoxy-CTP and
the RTS RadPrime DNA labeling system (Life Technologies, Inc.), and was hybridized to blots overnight at 42 C using
2 x 107 cpm labeled probe. The hybridized
blots were washed and exposed overnight at -70 C to Kodak
XAR film. The films were developed, and the signals were measured using
an Arcus II (Agfa, Wilmington, MA) scanner and the NIH Image
1.61 program. After stripping the blots, they were rehybridized to a
radiolabeled cDNA probe corresponding to 18S ribosomal RNA (rRNA) to
monitor total RNA loading.
Northern blot of LH/hCG receptor mRNA
When LH/hCG receptor mRNA expression was examined, Northern
blots were performed using a procedure identical to that described for
SR-BI mRNA. The cDNA probe used was a 750-mer probe from the
carboxyl-terminal region as reported previously from our laboratory
(25).
In situ hybridization
Ovaries from 25-day-old rats not subjected to any treatments
were collected and frozen in OCT compound. The frozen ovaries were cut
at -20 C, and 10-µm sections were mounted on silane-coated slides.
Tissue sections were fixed in 4% paraformaldehyde (pH 7.4), washed in
PBS, incubated with proteinase K (1 µg/ml), and rinsed with distilled
water. Subsequently, slides were placed in 0.1 M
triethanolamine (pH 8.0) and, after the addition of acetic anhydride
(final concentration, 0.25%, vol/vol), were incubated for 10 min.
Sections were washed with 2 x SSC (0.15 M NaCl and
0.015 M sodium citrate), dehydrated in graded alcohols
(50–100%), and dried. Antisense
[35S]UTP-labeled RNA probe was synthesized from
the 308-bp SR-BI cDNA template. The radiolabeled RNA probe was applied
to tissue sections, coverslips were overlaid and sealed with rubber
cement, and slides were incubated at 55 C overnight in a moist chamber.
After hybridization, sections were washed in 2 x SSC, treated
with ribonuclease A and ribonuclease T1, and subsequently washed in
increasingly lower concentrations of SSC. Sections were dehydrated
through graded alcohols and dried. The slides were processed for liquid
emulsion autoradiography using NTB-2 emulsion (Kodak).
Slides were developed after 2–4 days of exposure and counterstained
with hematoxylin-eosin.
Isolation of theca-interstitial cells
The general conditions were similar to those described by
Magoffin and Erickson (21) and Foghi et al.
(26). The procedure is briefly outlined as follows.
Twenty-five-day-old female Sprague Dawley rats (n = 20)
were killed by CO2 asphyxiation, and the ovaries
were collected in medium 199 containing 25 mM
HEPES (pH 7.4), 2 mM
L-glutamine, and 1 mg/ml BSA. The ovaries were
punctured with a needle to remove follicular fluid, cut into four to
six pieces, washed, and incubated for 90 min at 37 C in medium
containing collagenase (2.5 mg/ml) and DNase (10 µg/ml).
The details of the procedure were similar to those previously reported
from our laboratory (4). The dispersed cells were
centrifuged at 250 x g for 5 min and washed twice, and
the final pellet was suspended in a known volume. The suspended cells
were subjected to a single 5-min unit gravity purification. The
purified theca interstitial cells were washed twice and resuspended in
McCoy’s 5A medium. Viability was checked by trypan blue exclusion. The
dispersed cells were cultured in 60-mm tissue culture plates at a
density of 4 x 106 cells/dish for RNA
extraction. For cholesterol determination, the cells were placed in
35-mm tissue culture plates at a density of 1 x
106 cells/dish.
Cell culture
Theca-interstitial cells were cultured in McCoy’s 5A medium
containing 2 mM L-glutamine, 1 mg/ml BSA, 100
U/ml penicillin, and 100 µg/ml streptomycin, with or without other
test substances. The cells were cultured for 2, 4, or 6 days in a
humidified atmosphere of 95% air/5% CO2 at 37 C
with medium changed at 2-day intervals. For dose-response studies,
cells were cultured for 4 days with insulin and/or hCG at the
concentrations indicated in the figure legends. Total RNA was isolated
from the theca-interstitial cell cultures using TRIzol reagent
(Life Technologies, Inc.), and Northern blot analysis was
performed using radiolabeled cDNA probes for SR-B1, hCG receptor, and
18S mRNA as described above.
Cholesterol analysis in cultured theca-interstitial cells
using HPLC
As the amount of cholesterol extracted from the
theca-interstitial cell cultures was insufficient for quantitation
using the enzymatic assay, an alternate method using reverse phase HPLC
was employed. The theca-interstitial cell cultures were treated with 10
µg/ml insulin, 100 ng/ml hCG, or 10 µg/ml insulin plus 100 ng/ml
hCG and incubated for 4 days. At the end of the incubation period,
cells were solubilized in 1 ml 0.5 N NaOH. The cell lysate
was extracted with 1 ml hexane-isopropanol (3:2, vol/vol) followed by
two extractions with 1 ml hexane. Extraction efficiency was determined
by the addition of tracer amounts of
[14C]cholesterol and
[3H]cholesteryl oleate (Amersham Pharmacia Biotech) The extraction efficiency for cholesterol was
86.0 ± 2%, and that for cholesteryl esters was 82.5 ± 3%.
The pooled extracts were dried under N2,
dissolved in hexane, divided into two equal aliquots (free cholesterol
and total cholesterol), and dried under N2. The
samples for total cholesterol were saponified at 80 C for 1 h in
1.78 N KOH in 90% ethanol. Samples were diluted with an
equal volume of water, the lipids were extracted three times with 1 ml
hexane, and the pooled extracts were dried under
N2. Cholesterol was measured by HPLC analysis
(Waters Corp., Milford, MA) with a Waters Nova-Pak
C18 reverse phase column and a mobile phase
consisting of acetonitrile/0.1% trifluoroacetic acid (95:5) at a flow
rate of 1.0 ml/min. Detection of the product was at 205 mm with a
Waters Corp. model 490E programmable wavelength detector
in combination with a Waters Corp. model 600 controlling
unit and model 710 autoinjector, all operated by the Millennium
Software Package (Waters Corp., Milford, MA). The
standard curve was generated using authentic cholesterol as
standard.
Statistical analysis
The statistical analysis was initially carried out using ANOVA.
If ANOVA indicated significant differences within the dataset, pairwise
comparisons were performed using unpaired Student’s t test.
Each experiment was repeated at least three times with similar results,
and the data presented are the results of a single experiment.
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Results |
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. An intense
hybridization signal in the theca interna as well as in the stroma
cells was seen in the darkfield photomicrographs.
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show that treatment with insulin alone caused an elevation in total
cellular cholesterol content after 12 h of treatment. At shorter
time intervals, the increase was less than that seen at 12 h.
Administration of hCG caused a marked decline in the total cellular
cholesterol levels, and the decrease was noticeable as early as 4
h after treatment. The decline in cholesterol content was maximum at
12 h. When insulin administration was followed by hCG, the decline
in total cellular cholesterol was even more pronounced than that seen
with hCG treatment alone. The cholesterol content in the control
ovaries remained unchanged over the course of treatment. Serum
progesterone increased in hCG-treated rats at all time intervals
examined (data not presented). The depletion of total cholesterol was
more pronounced when insulin treatment was followed by hCG, indicating
that insulin augments hCG stimulation of steroidogenesis. Free
cholesterol measurements also followed a pattern similar to that seen
with total cholesterol (Fig. 3). Because
granulosa cells were removed before cholesterol determination, the
changes in cholesterol content are attributable to theca-interstitial
cells. These studies show that insulin increased the cellular
accumulation of cholesterol.
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. hCG
administration increased SR-BI mRNA expression significantly over the
control level. Insulin administration also increased SR-BI mRNA
expression, but the extent of stimulation was less than that produced
by hCG treatment. The combination of insulin and hCG produced an
increased response, higher than that produced by either treatment
alone. To normalize SRBI mRNA expression for RNA loading, the SR-BI
mRNA blot was stripped and rehybridized with a cDNA corresponding to
18S rRNA (Fig. 4B). The densitometric scan of the SR-BI mRNA
of each blot normalized for the 18S rRNA is shown in Fig. 4C. These
results show that insulin and hCG stimulate the expression of SR-BI
mRNA in theca-interstitial cells. Additionally, the combination of
insulin and hCG produced an additive effect.
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show that treatment with insulin
alone caused an increase in the hybridization signal on days 2, 4, and
6. Treatment with hCG alone, however, did not cause an increase in
SR-BI expression on day 2, and the stimulation was marginal on days 4
and 6. This is in contrast to the in vivo situation, where
hCG administration stimulated the expression of SR-BI mRNA (Fig. 4
).
However, in the presence of insulin, hCG exerted a stimulatory effect
on SR-BI mRNA expression in vitro at all time intervals
examined.
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was
stripped and hybridized with a 750-bp
32P-labeled LH/hCG receptor cDNA probe
(25). Figure 6 shows the
expression of LH/hCG receptor mRNA in different treatment groups.
The results show that inclusion of insulin helps maintain the receptor
for LH/hCG in cultured theca-interstitial cells and enhances the
expression of LH/hCG receptor mRNA on days 2 and 4. However, the
stimulation was diminished by day 6. Even in the presence of insulin,
LH/hCG receptor mRNA showed decreased expression by day 6. From these
experiments we conclude that insulin, in addition to stimulating SR-BI
mRNA expression, has a protective effect on LH/hCG receptor mRNA.
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show that insulin treatment alone produced a stimulatory effect on the
expression of SR-BI mRNA, and a modest increase was seen with increased
insulin concentrations. Cultures incubated in the presence of insulin
and hCG produced a greater stimulation of SR-BI mRNA expression. These
data confirm the potentiating roles of hCG and insulin on the
stimulation of SR-BI mRNA expression. The stimulatory effect of
increasing concentrations of hCG in the presence of 10 µg/ml insulin
was then tested. The results presented in Fig. 8 show that insulin caused an increase in
SR-B1 mRNA expression. Increasing concentrations of hCG in the absence
of insulin produced no stimulatory effect due to the reasons explained
above. In the presence of insulin, hCG produced a stimulatory effect on
SR-BI mRNA expression, and the responsiveness was increased further
with increased hCG concentration.
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show that treatment
with insulin alone resulted in a slight, but statistically
insignificant, increase in total and esterified cholesterol contents.
Treatment with hCG alone did not produce a significant change in the
content of total or esterified cholesterol. This lack of hCG response
in vitro is consistent with the inability of hCG to induce
SR-BI mRNA expression in vitro, possibly due to the loss of
hCG receptors, as described above. In cultures treated concomitantly
with hCG and insulin, there was a significant depletion in the contents
of total and esterified cholesterol. This is in agreement with the
in vivo studies in which depletion of cholesterol content in
the ovary was observed in response to hCG and insulin. From the
in vivo and in vitro studies, it is clear that
HDL receptor in the theca-interstitial cells is appreciably increased
in response to the combined action of insulin and hCG over that
produced by either agent alone.
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Discussion |
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Steroidogenic cells take up cholesterol from both LDL as well as HDL (14, 15, 18). The uptake of cholesterol from LDL proceeds by a mechanism well defined by Brown and Goldstein involving receptor-mediated endocytotic internalization followed by lysosomal hydrolysis (17). The uptake of cholesterol from HDL is also a receptor-mediated process, but the mechanism of transfer of cholesterol from the cell membrane is believed to be a selective transfer, and this process is different from the LDL-mediated cholesterol transfer (27, 28). The receptor for HDL in the ovarian tissues was initially identified by ligand binding studies (12, 14, 16, 29, 30). More recently, it was shown that a previously cloned protein, SR-BI, meets the criteria for serving as the HDL receptor, and its presence has been confirmed in ovarian tissue by Northern blotting, Western blotting, and in situ hybridization histochemistry (19, 20, 31, 32, 33).
The present studies clearly show that insulin increased the expression of SR-BI mRNA in theca-interstitial cells under both in vivo and in vitro conditions. Although insulin by itself had minimal effect on steroidogenesis, theca-interstitial cells exposed to insulin showed in an increase in the intracellular accumulation of cholesterol. This increase is due to increased transport of cholesterol into theca-interstitial cells mediated by an increased expression of SR-BI mRNA. When theca-interstitial cells were exposed to hCG in vivo, SR-BI mRNA expression was induced. The extent of induction of SR-BI mRNA expression in response to a combination of insulin and hCG produced an even higher magnitude of SR-BI mRNA expression. Examination of cellular cholesterol accumulation revealed a different, but expected, picture.
Although insulin treatment alone produced an increase in the intracellular cholesterol accumulation, hCG treatment produced a depletion of cellular cholesterol. A depletion is expected, as hCG treatment increases the utilization of cellular cholesterol as a result of increased conversion to steroids. A combination of hCG and insulin produced an even larger depletion in cellular cholesterol, perhaps due to the potentiating effect of insulin on hCG-mediated steroidogenesis. This is in keeping with the previous studies in which a potentiating effect of insulin on theca-interstitial cell androgen production has been reported (6). The failure of hCG to produce a stimulatory effect on SR-BI mRNA expression under in vitro conditions is caused by the loss of hCG receptors from the cultured theca-interstitial cells due to either down-regulation of the receptors or the inability of the cultured cells to maintain LH/hCG receptors. Another possibility is that any contaminating protease activity of collagenase may have damaged cell surface LH/hCG receptors through proteolysis. The loss of LH/hCG receptors from the cultured cells was confirmed by Northern blot analysis. When theca-interstitial cells were cultured in the presence of insulin and hCG, the LH/hCG receptors were maintained by these cells, and this was confirmed by Northern blot analysis. Under these conditions, a combination of hCG and insulin produced an increase in the expression of SRB-I mRNA and a reduction in the intracellular level of cholesterol. These results clearly show that insulin has a stimulatory effect on the transport of cholesterol to theca-interstitial cells, which is preceded by an increase in the expression of the HDL receptor, SR-BI.
The mechanism of induction of SR-BI mRNA expression by insulin is not understood. It appears that as insulin treatment alone did not cause a depletion of endogenous cholesterol, the mechanism of insulin induction of SR-BI mRNA expression may be different from that seen in response to the induction produced by hCG treatment. The current dogma of the induction of cholesterol-responsive genes is that depletion of the cellular cholesterol pool results in activation of a cysteine protease. The proteolytic cleavage of a precursor protein generates a transcription factor known as sterol response element-binding protein (34). This is followed by translocation of sterol response element-binding protein to the nuclei, where it interacts with sterol response element to stimulate the transcription of cholesterol-responsive genes, including LDL receptor (35). In the endocrine cells, SR-BI mRNA expression is expected to follow a similar pathway, because these cells import, rather than export, cholesterol. Insulin treatment, on the other hand, does not lead to depletion of cellular cholesterol. Therefore, we speculate that the mechanism of induction of SR-BI expression by insulin might follow a mechanism different from that described above.
The present studies also show the ability of insulin-treated theca-interstitial cells to respond to hCG under in vitro conditions. The ability of IGF-I to induce the expression of LH receptor mRNA in theca-interstitial cells has been previously reported (36). Whether insulin acts as a pleiotropic agent that helps maintain cultured cells in a metabolically healthy environment or induces the synthesis of LH/hCG receptors is not currently understood. Further studies are required to assess the mechanism involved in the insulin-mediated rescue of LH/hCG receptors in cultured theca-interstitial cells. The present study also suggests that insulin is capable of increasing the transport of cholesterol into theca-interstitial cells. The increased accumulation of cholesterol in theca-interstitial cells is secondary to an increase in the expression of SR-BI mRNA. In addition, insulin potentiates the hCG-mediated induction of SR-BI mRNA in these cells. Thus, insulin acts in theca-interstitial cells by two different mechanisms. First, it increases the expression of SR-BI mRNA, thereby facilitating cholesterol transport. Second, insulin increases LH/hCG receptor mRNA expression. Both of these processes lead to an increase in steroid biosynthesis in theca-interstitial cells. It is speculated that under hyperinsulinemic states, increased cholesterol transport to theca-interstitial cells is one of the mechanisms responsible for the overproduction of ovarian androgens. Although the insulin/IGF-I system serves as a physiological regulator of ovarian function, pathological conditions that lead to the overproduction of insulin and/or IGF-I could cause hypersecretion of androgens. Based on the present data, it is concluded that increased cholesterol transport into theca-interstitial cells might be one of the factors that contribute to overproduction of androgens by the ovary.
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Footnotes |
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Received June 12, 2000.
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