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From the * Division of Hormone Research,
Department of Cell Biology, Georgetown University School of Medicine,
Washington, DC; and the Division of
Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns
Hopkins University School of Public Health, Baltimore, Maryland.
| Correspondence to: Dr Barry R. Zirkin, Department of Biochemistry and Molecular Biology, John Hopkins University School of Public Health, 615 N Wolfe St, Baltimore, MD 21205-2179 (e-mail: brzirkin{at}jhsph.edu ). |
| Received for publication September 5, 2001; accepted for publication January 7, 2002. |
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Abstract |
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Key words: Mitochondria, steroidogenic acute regulatory protein
The Brown Norway rat presents an age-related primary testicular deficit similar to that observed in the human (Zirkin and Chen, 2000). In this rat, Leydig cell numbers do not change with aging. Rather, the capacity of old Leydig cells to produce testosterone is reduced (Chen et al, 1994, 1996; Luo et al, 1998; Zirkin and Chen, 2000). A decrease in Leydig cell steroidogenesis could result from a reduction in any of the enzymatic activities involved in testosterone synthesis (Hall, 1984, 1998) or could reflect a decrease in the amount of substrate available for these enzymes to form testosterone. Previous studies have shown that all enzymes involved in the synthesis of testosterone are decreased with aging (Luo et al, 1996, 2001). More recently, we reported that both the side-chain cleavage cytochrome P450scc, responsible for the first step of steroidogenesis (Hall, 1984), and the steroidogenic acute regulatory protein (StAR), a hormone-induced protein considered to be involved in mediating cholesterol transport into the mitochondria (Clark et al, 1994), were decreased in Leydig cells from old Brown Norway rats compared to young rats (Luo et al, 2001). However, hormonal stimulation resulted in increases of testosterone production and StAR protein expression in cells from both old and young rats, indicating that the old Leydig cells have not lost the ability to respond to hormones (Luo et al, 2001). This further suggested that the net testosterone decrease observed might have additional causes, such as a deficit in the basal/unstimulated pool of cholesterol available for steroidogenesis.
Since the limiting step of the steroidogenic cascade has been shown to be the transport of cholesterol to P450scc, the first enzyme of the cascade (Crivello and Jefcoate, 1980; Privalle et al, 1983), it is easy to envision that the alteration of either the pool of cholesterol involved in this process or the intramitochondrial transport of cholesterol could perturb the steroidogenic process. In the present study, we compared the size of the mitochondrial cholesterol pool available for pregnenolone formation by young and old cells, the steroidogenenic response of the cells in the presence of nonlimiting levels of cholesterol, and the expression of peripheral benzodiazepine receptor (PBR), a high-affinity mitochondrial cholesterol-binding protein (Lacapère et al, 2001; Li et al, 2001) also known to mediate cholesterol transport in steroidogenic tissues (Krueger and Papadopoulos, 1990; Papadopoulos, 1993; Li and Papadopoulos, 1998). We report that aging is associated with decreases in the cholesterol stores available for steroidogenesis and in the expression of PBR protein, suggesting that these 2 events contribute to the testosterone decrease observed in aging.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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,5-epoxy-17-hydroxy-3-keto-5-androstan-2-carbonitrile)
was a gift from Stegram Pharmaceuticals (Sussex, United Kingdom). SU-10603, an
inhibitor of P450c17 enzyme, was a gift from CIBA-GEIGY (Suffern, NY).
Purified human chorionic gonadotropin (hCG; batch CR-125 of biological potency
11 900 IU/mg) was a gift from Dr A. F. Parlow, National Institute of Diabetes
and Digestive and Kidney Diseases (NIDDK) National Hormone & Pituitary
Program at the National Institutes of Health (Bethesda, Md). Antibodies to
pregnenolone and testosterone were obtained from ICN Pharmaceuticals Inc
(Costa Mesa, Calif). The antibody to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was from Trevigen (Gaithersburg, Md). Cell culture supplies were
purchased from Life Technologies Inc (Grand Island, NY). Percoll was purchased
from Pharmacia (Piscataway, NJ). All other chemicals were of analytical
quality and were obtained from various commercial sources.
Animals
Adult male Brown Norway rats aged 4 (young) or 24 (old) months were
obtained through the National Institute on Aging, supplied by Harlan
Sprague-Dawley Inc (Indianapolis, Ind), and housed in controlled light and
temperature conditions as previously described
(Luo et al, 2001).
Isolation of Leydig Cells
Leydig cells from young and old rats were isolated and purified by Percoll
gradient and, in some studies, by centrifugal elutriation and Percoll gradient
(Klinefelter et al, 1987). The
purity of the cells, assessed by staining for 3ß-hydroxysteroid
dehydrogenase, was about 85% after Percoll gradient alone and was greater than
93% after a combination of Percoll gradient and centrifugal elutriation. This
was true for both young and old cells. Cells from pooled testes from up to 5
rats were purified as 1 individual sample.
Mitochondrial Preparation
Mitochondria were prepared as previously described
(Krueger and Papadopoulos,
1990). All steps of the procedure were done at 4°C. Briefly,
aliquots of purified Leydig cells were washed in phosphate-buffered saline
(PBS), resuspended in buffer A (50 mM Tris-HCl [pH 7.2], 250 mM sucrose), and
homogenized with an electric Teflon-glass homogenizer. The homogenate was
centrifuged at 800 x g for 10 minutes, the supernatant was
collected, and the pellet was homogenized and centrifuged again. The 2
supernatants were pooled and centrifuged at 10 000 x g for 15
minutes. The resulting mitochondrial pellet was washed twice in buffer B (10
mM potassium phosphate [pH 7.4], 0.25 M sucrose, 5 mM MgCl2, 20 mM KCl, and 15
mM triethanolamine-HCl) containing 5 µM trilostane, which, by inhibiting
3ß-hydroxysteroid dehydrogenase, prevents pregnenolone metabolism
(Potts et al, 1978). The
mitochondrial preparations were assayed for protein concentration and were
used in steroidogenesis and PBR binding experiments. Each mitochondrial sample
was prepared from Leydig cells obtained from the pooled testes of 2-5 rats in
order to obtain purified mitochondrial aliquots containing at least 500 µg
protein/mL.
Intact Cell Steroidogenesis
Leydig cells were suspended in ice-cold Dulbecco modified Eagle medium
supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100
µg/mL streptomycin, and they were then incubated for 2 hours with the
agents to be tested in a 37°C shaking water bath, in the presence or
absence of 10 µM trilostane plus 5 µM SU-10603
(Gower, 1974). The reactions
were stopped by freezing the samples at -20°C for later determination of
pregnenolone or testosterone concentration by radioimmunoassay.
Measurement of Cholesterol Accumulation Upon Hormonal
Stimulation
In these experiments, intact Leydig cells were initially incubated for 2
hours at 37°C in the presence of 500 µM AMG, a specific inhibitor of
P450scc, and in the presence or absence of 50 ng/mL hCG, according
to a protocol previously described (Boujrad
et al, 1994). The AMG-mediated blocking of cholesterol metabolism
upstream of the P450scc reaction results in the accumulation of
cholesterol in the mitochondrial membranes, including the basal pools and
those transferred to the mitochondria upon hormonal treatment. The cells were
then washed twice with ice-cold PBS containing AMG and further processed for
mitochondrial preparations as described above, except that AMG was included in
every step until the last centrifugation. At this stage, AMG was removed and,
after a final wash of the pellet in AMG-free buffer, the mitochondria were
resuspended and incubated for 20 minutes at 37°C in the presence of 10
µM trilostane, 15 mM isocitric acid, and 0.5 mM NADP in order to allow the
P450scc to function. Steroids were then extracted and measured by
radioimmunoassay. The difference in pregnenolone formed between the aliquots
from cells incubated with or without hCG corresponds to the cholesterol that
was accumulated during stimulation and is proportional to the cholesterol
transport.
Measurement of Mitochondrial Pregnenolone Synthesis
Mitochondria were resuspended in buffer B at a final concentration of
0.5-1.0 mg/mL of protein. In some experiments, 22R-hydroxycholesterol was
added to the samples. The reaction was started by the addition of isocitric
acid and NADP (at 15- and 0.5-mM final concentrations, respectively) in a
final volume of 250 µL. After a 20-minute incubation at 37°C, the
reaction was stopped by adding 100 µL of ethanol containing 2000 counts/min
(cpm) of [3H] pregnenolone as a recovery marker, followed by 1 mL
of diethyl ether. After extraction, the organic phase was collected and
evaporated to dryness. Pregnenolone was measured by a specific
radioimmunoassay.
Radioimmunoassays
Pregnenolone and testosterone productions were measured using specific
radioimmunoassays as previously described
(Papadopoulos et al, 1990; Boujrad et al, 1994), following
the conditions recommended by the supplier of the antibodies. The recoveries
were determined and used to correct the results. Analysis of the
radioimmunoassay data was performed using the MultiCalc Software from EG &
G Wallac Inc (Gaithersburg, Md).
Radioligand-Binding Assays
The binding experiments were performed as previously described
(Papadopoulos et al, 1990). Briefly, each individual sample of Leydig cells was purified from the pooled
testes of 3 rats, either young or old, and 3 independent sets of such pools
were used in each experiment. The purified cells were washed twice with
ice-cold PBS and homogenized in PBS with a Teflon-glass homogenizer. Aliquots
of cell lysate containing 20 µg protein/sample were incubated at 4°C in
the presence of 0.03-20 nM [3H]PK 11195 in a final incubation
volume of 0.3 mL. Nonspecific binding was determined by adding 200-fold excess
of unlabeled ligand in some samples. After a 90-minute incubation, assays were
stopped by filtration through Whatman GF/B filters (Brandel, Gaithersburg, Md)
equilibrated in 0.1% polyethyleneimine and washed with 20 mL ice-cold PBS.
Radioactivity trapped on the filters was determined by liquid scintillation
counting. The dissociation constant (Kd) and the number of binding sites
(Bmax) were determined by Scatchard plot analysis of the saturation isotherms
generated using the LIGAND program (KELL, version 4.0, Biosoft Inc, Ferguson,
Mo) (Munson and Rodbard,
1980).
Immunoblot (Western) Analysis
Whole cells or mitochondria were solubilized in Laemmli buffer, and the
proteins, loaded either at equal cell number per lane or equal protein amount
per lane, were fractionated by one-dimensional sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred
onto nitrocellulose, as previously described
(Li et al, 2001). Immunoblot
analysis of membranes was performed using a rabbit anti-PBR antiserum prepared
by sequential immunization with a peptide of PBR protein (conserved sequence)
coupled to keyhole limpet hemocyanin (1:1000) and a goat anti-rabbit
immunoglobulin G conjugated with horseradish peroxidase (1:5000)
(Li et al, 2001). Positive
bands were then detected by chemiluminescence with the Renaissance kit
(DuPont/NEN). Equal protein loading was assessed by reprobing the blots with
an anti-GAPDH antiserum (1:1000; Trevigen). Densitometric analysis of the
immunoreactive protein bands was performed using OptiQuant Software from
Packard Bioscience (Meriden, Conn).
RNA Blot (Northern) Analysis
Total RNA was isolated from freshly isolated young or old Leydig cells as
previously described (Chomczynski and
Sacchi, 1987; Luo et al,
2001). The RNA extraction was carried out in the presence of a
known amount of 35S-labeled RNA as an internal standard, and the
results were normalized for the variations of RNA recovery. Equal numbers of
young and old cells were loaded onto denaturing 1.2% agarose gels and
electrophoresed. The gels were blotted by capillary transfer onto a nylon
membrane and were further hybridized with a 32P-labeled
complementary DNA (cDNA) probe for PBR. The cDNA of the full-length mouse PBR
was labeled with deoxycytidine 5'-[-32P] triphosphate
(dCTP) at ca 108 cpm/µg DNA using a random primer synthesis
method.
Protein Measurement
Protein levels were measured by the Bradford method
(Bradford, 1976) using the
Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, Calif) with BSA as
a standard.
Statistical Analysis
Results are presented as mean plus or minus standard error of the mean of
up to 4 independent experiments. To determine if the means of specific
parameters differed significantly between young and old rats, they were
compared with unpaired t tests using the InStat program (version 3.0)
from GraphPad Software, Inc (San Diego, Calif). The Kd and Bmax values for PBR
were compared between young and old rats with the Mann-Whitney U
test, and multiple groups were compared by one-way analysis of variance, both
using InStat. The changes observed with aging were considered statistically
significant for P less than.05.
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Results |
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In some experiments, cells were incubated in the presence of trilostane and SU-10603 to inhibit pregnenolone metabolism, in order to examine the P450scc activity by itself instead of the whole steroidogenic cascade. The difference in the levels of pregnenolone produced between old and young cells was similar to that found for testosterone produced in the absence of the inhibitors (data not shown).
Changes in Mitochondrial Cholesterol Accumulation With Aging
Since the availability of cholesterol appeared to be a critical factor in
old cells, we performed further experiments to indirectly measure the size of
the hormonally induced cholesterol pool. To this end, cells were incubated
with hCG and the inhibitor of P450scc, AMG, and mitochondria were
then isolated from these cells in the presence of AMG. As shown in
Figure 2, when young Leydig
cells were incubated with AMG and hCG, there was a four- to sixfold increase
in pregnenolone synthesis upon removal of the inhibitor from the mitochondria
isolated from these cells, indicating that hCG stimulates an active
accumulation of cholesterol into mitochondria. The same paradigm used on
mitochondria from old rats showed that the hCG-induced pool of cholesterol
available for P450scc after AMG removal was smaller than in young
cells; there was only a two- to threefold increase in pregnenolone synthesis
between mitochondria from control and hCG-treated old cells. This decrease in
steroid formation in old vs young cells indicated that the hormonally
activated cholesterol transport or loading to mitochondria was reduced by
80%-90% in old vs young cells when expressed as net steroid formed after
subtracting the basal levels. It should also be noted that the basal levels of
steroid formation were greatly diminished, by 60%-90%, in the mitochondria
from old vs young cells, indicating an impairment of the homeostasis of
cholesterol in the mitochondria of old rats.
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Age-Dependent Changes in PBR Messenger RNA and Protein
Expression
Considering the results presented above, showing that both basal and
hormone-regulated levels of cholesterol were lower in old than in young cells,
we compared the expression of the PBR, a mitochondrial protein known to
mediate cholesterol transport in steroidogenic tissues, in young and old rat
Leydig cells. As shown in Figure
3, PBR messenger RNA (mRNA) expression per cell was significantly
lower (30% decrease) in Leydig cells from old than from young rats. For this
study, total RNA from equal numbers of young and old cells was applied per
lane. Aging also was accompanied by a significant decrease in the expression
of the PBR protein (Figure 4).
For this study, equal amounts of protein were applied per lane, and the
Western blots were reprobed for GAPDH as a loading control. Thus, both PBR
mRNA and protein expression in Leydig cells were affected by aging.
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Age-Induced Changes in PBR Binding Sites
Radioligand-binding assays were performed on cell lysates from young and
old Leydig cells in order to evaluate if aging has an effect on PBR expression
and function. As shown in Figure
5A, the Kd of PBR was significantly decreased by 50% (P
<.05) in old vs young cells. This increase of affinity of the receptor
could be due to changes in protein structure or to changes in the environment
of the receptor affecting its folding and conformation. Bmax values were also
decreased (Figure 5B), but to a
lesser extent, in old vs young cells. However, the changes in PBR protein
could be greater than shown by the Bmax values. These values are expressed per
milligram of total protein, and proteins other than PBR, such as the
steroidogenic enzymes, are known to decrease with aging. Thus, if a large
number of proteins decrease significantly, the proportion represented by PBR
could become larger, even though PBR itself might be decreased. Indeed, as
shown by immunoblot analysis, the decrease in Bmax was less pronounced than
the decrease found in the protein itself, indicating that age-related changes
in the mitochondrial membrane may slightly improve the access of the ligand to
the receptor, attenuating the decrease in the Bmax compared to the whole PBR
protein.
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Discussion |
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The transport of cholesterol to the inner mitochondrial membrane is considered to be the rate-limiting step in steroidogenesis (Jefcoate et al, 1974; Hall, 1984). If the cholesterol available for translocation to the P450scc enzyme was reduced in old cells or if cholesterol movement to the P450scc enzyme was impeded, reduced testosterone production by the Leydig cells would be the likely outcome. We hypothesized that a reduction in cholesterol stores, or deficits in cholesterol transport, might indeed explain age-related decreases in testosterone production by the Leydig cells. To test this hypothesis, we first compared the steroidogenic capacity of young and old Leydig cells by measuring testosterone production by cells incubated with hCG or 22R-hydroxycholesterol, the latter a hydrosoluble substrate that easily enters mitochondria and thus bypasses the rate-limiting step of cholesterol transport between mitochondrial membranes (Hall, 1998). In both cases, the levels of testosterone produced by the old cells were significantly reduced compared to young cells. Interestingly, however, testosterone production in the presence of 22R-hydroxycholesterol was dramatically increased in comparison to that stimulated by hCG. This was true of both young and old cells. Moreover, though the absolute amounts differed, the fold-increase in testosterone production from basal levels was at least equivalent in young and old cells. The difference observed in steroid production between young and old cells incubated with 22R-hydroxycholesterol suggests that the steroidogenic enzyme content per cell in the old cells is reduced and that this limits testosterone production. Indeed, previous studies have suggested that this is the case (Luo et al, 1996; Zirkin and Chen, 2000). The observation that the mitochondria of old cells produce less pregnenolone than those of young cells indicates that P450scc, in particular, is reduced. However, given that the fold-increase in testosterone production was at least equivalent in young and old cells, it seems clear that the individual molecules of cytochrome P450scc function normally in old cells when nonlimiting amounts of substrate are present.
These observations led us to hypothesize that aging might be accompanied by a defect in cholesterol transport to mitochondria, resulting in less cholesterol available to the cytochrome P450scc in old cells. To test this hypothesis, Leydig cells were incubated with hCG and the P450scc inhibitor AMG, leading to the accumulation of hormonally recruited cholesterol into mitochondrial membranes (Boujrad et al, 1994). This paradigm allows for an indirect quantification of the cholesterol stores used in steroid synthesis, since the measurement of mitochondrial steroidogenesis after AMG removal is proportional to the cholesterol pool available for P450scc. This experiment revealed a significant decrease in mitochondrial steroidogenesis in old vs young rats that was far greater than could be accounted for by a decrease in cytochrome P450scc. These results indicated that, as hypothesized, there is a reduction in the amount of cholesterol transported into the mitochondria of old cells upon hormonal stimulation of the cells. Indeed, even without hormonal stimulation, significant differences were observed in steroid production between young and old cells. Taken together, these results suggest a potential defect in cholesterol transport in old Leydig cells.
The mechanism by which cholesterol translocation occurs continues to be debated, although it now seems clear that both StAR (Clark et al, 1994, 1995; Stocco and Clark, 1996) and PBR (Papadopoulos, 1993; Papadopoulos et al, 1997a,b) are involved. PBR, an integral outer mitochondrial membrane protein involved in the transport of cholesterol from the outer to the inner mitochondrial membranes in steroidogenic tissues (Krueger and Papadopoulos, 1990; Li and Papadopoulos, 1998), was recently shown to be a high-affinity cholesterol-binding protein (Lacapère et al, 2001; Li et al, 2001). Decreases in PBR expression have been shown in various models to correlate with decreases in steroid synthesis (Papadopoulos et al, 1997a,b). PBR is modified rapidly upon hormonal stimulation (Boujrad et al, 1994, 1996) and thus appears to play a role in the hormone-mediated response as well as in the maintenance of the basal pool of mitochondrial cholesterol (Papadopoulos et al, 1990).
In the present study, we found that both PBR mRNA and protein expression were reduced in old compared to young Leydig cells. Moreover, the receptor-binding experiments showed that both the Kd and Bmax of PBR were decreased in old cells. The increased affinity of the receptor could be due to changes in the protein structure, as well as to changes in the environment of the receptor affecting its folding and conformation. Although the ligand-biding site and the cholesterol-binding motif of PBR are localized on different domains of the receptor (Li and Papadopoulos, 1998; Li et al, 2001), a structural change in one area would most probably have consequences in other parts of the protein. Similarly, changes in the membrane environment adjacent to PBR would not only affect the ligand-binding site, but also other functional domains such as the cholesterol-binding site. Considering that PBR mediates the transfer of cholesterol from the outer to the inner mitochondrial membranes in steroidogenic cells (Krueger and Papadopoulos, 1990), any deficit in its expression/function should lead to a decrease in the cholesterol transport, and consequently, the cholesterol pool used during steroid synthesis. Indeed, the experiments performed in the presence of AMG confirmed the existence of a defecit in the basal and hormone-induced pools of cholesterol in mitochondria from old Leydig cells, which correlates with the decrease observed in PBR expression.
In conclusion, the results of the present study suggest that decreases in cholesterol transport to the mitochondria and changes in PBR may play significant roles in the decline of testosterone observed in aging. It now seems evident that a variety of factors are likely to be involved in age-related decreases in steroidogenesis, including a reduction in PBR and StAR and a reduction of the steroidogenic enzymes sequestered in the mitochondria and smooth endoplasmic reticulum. In this context, an association of PBR and StAR fusion proteins has recently been shown at the level of the mitochondrial membrane in a reconstituted cell model (West et al, 2001). Taken together, these observations suggest that correcting any one of these factors therapeutically may lead to an improvement in testosterone production but not a total recovery of the levels of testosterone present in young individuals.
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Acknowledgments |
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Footnotes |
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bonavera JJ, Swerdloff RS, Leung A, Leu YH, Baravarian S, Superlano
L, Sinha-Hikim AP, Wang C. In the male brown Norway (BN) male rat,
reproductive aging is associated with decreased LH pulse amplitude and area.
J Androl. 1997;18:
359
-365.
Boujrad N, Gaillard J-L, Garnier M, Papadopoulos V. Acute action of choriogonadotropin in Leydig tumor cells. Induction of a higher affinity benzodiazepine receptor related to steroid biosynthesis. Endocrinology. 1994; 135: 1576 -1583.[Abstract]
Boujrad N, Vidic B, Papadopoulos V. Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Endocrinology. 1996; 137: 5727 -5730.[Abstract]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem. 1976; 72: 248 -254.[Medline]
Chen H, Hardy MP, Huhtaniemi I, Zirkin BR. Age-related decreased
Leydig cell testosterone production in the Brown Norway rat. J
Androl. 1994;15:
551
-557.
Chen H, Huhtaniemi I, Zirkin BR. Depletion and repopulation of Leydig cells in the testes of aging Brown Norway rats. Endocrinology. 1996; 137: 3447 -3452.[Abstract]
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162: 156 -159.[Medline]
Clark BJ, Soo S-C, Caron KM, Ikeda Y, Parker KL, Stocco DM.
Hormonal and developmental regulation of the steroidogenic acute regulatory
protein. Mol Endocrinol.
1995; 9:
1346
-1355.
Clark BJ, Wells J, King SR, Stocco DM. The purification, cloning,
and expression of a novel luteinizing hormone-induced mitochondrial protein in
MA-10 mouse Leydig tumor cells. J Biol Chem.
1994; 269:
28314
-28322.
Crivello JF, Jefcoate CR. Intracellular movement of cholesterol in
rat adrenal cells. J Biol Chem.
1980; 255:
8144
-8151.
Falahati-Nini A, Riggs BL, Atkinson EJ, O'Fallon WM, Eastell R, Khosla S. Relative contributions of testosterone and estrogen in regulating bone resorption and formation in normal elderly men. J Clin Invest. 2000;106: 1553 -1560.[Medline]
Gower DB. Modifiers of steroid-hormone metabolism: a review of their chemistry, biochemistry and clinical applications. J Steroid Biochem. 1974;5: 501 -523.[Medline]
Gruenewald DA, Naai MA, Hess DL, Matsumoto AM. The Brown Norway rat as a model of male reproductive aging: evidence for both primary and secondary testicular failure. J Gerontol. 1994; 49: B42 -50.[Abstract]
Hall PF. Cellular organization for steroidogenesis. Int Rev Cytol. 1984;86: 53 -95.[Medline]
Hall PF. Testicular steroid synthesis: organization and regulation. In: Knobil E, Neil J, eds. The Physiology of Reproduction. New York: Raven Press; 1998: 975 -998.
Jankowska EA, Rogucka E, Medras M, Welon Z. Relationships between age-related changes of sex steroids, obesity and body fat distribution among healthy Polish males. Med Sci Monit. 2000; 6: 1159 -1164.[Medline]
Janssens H, Vanderschueren DM. Endocrinological aspects of aging in men: is hormone replacement of benefit? Eur J Obstet Gynecol Reprod Biol. 2000;92: 7 -12.[Medline]
Jefcoate CR, Simpson ER, Boyd GS. Spectral properties of rat adrenal mitochondrial cytochrome P-450. Eur J Biochem. 1974; 42: 539 -551.[Medline]
Kenny AM, Prestwood KM, Marcello KM, Raisz LG. Determinants of bone density in healthy older men with low testosterone levels. J Gerontol A Biol Sci Med Sci. 2000; 55: M492 -497.
Klinefelter GR, Hall PF, Ewing LL. Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod. 1987; 36: 769 -783.[Abstract]
Krueger KE, Papadopoulos V. Peripheral-type benzodiazepine
receptors mediate translocation of cholesterol from outer to inner
mitochondrial membranes in adrenocortical cells. J Biol
Chem. 1990;265:
15015
-15022.
Lacapère JJ, Delavoie F, Li H, Péranzi G, Maccario J, Papadopoulos V, Vidic B. Structural and functional study of reconstituted peripheral benzodiazepine receptor (PBR). Biochem Biophys Res Commun. 2001;284: 536 -641.[Medline]
Li H, Papadopoulos V. Peripheral-type benzodiazepine receptor
function in cholesterol transport. Identification of a putative cholesterol
recognition/interaction amino acid sequence and consensus pattern.
Endocrinology,
1998; 139:
4991
-4997.
Li H, Yao Z, Degenhardt B, Teper G, Papadopoulos V. Cholesterol
binding at the cholesterol recognition/interaction amino acid consensus (CRAC)
of the peripheral-type benzodiazepine receptor and inhibition of
steroidogenesis by an HIV TAT-CRAC peptide. Proc Natl Acad Sci
USA. 2001;98:
1267
-1272.
Luo L, Chen H, Stocco DM, Zirkin BR. Leydig cell protein synthesis
and steroidogenesis in response to acute stimulation by luteinizing hormone in
rats. Biol Reprod.
1998; 59:
263
-270.
Luo L, Chen H, Zirkin BR. Are Leydig cell steroidogenic enzymes
differentially regulated with aging? J Androl.
1996; 17:
509
-515.
Luo L, Chen H, Zirkin BR. Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. J Androl. 2001;22: 149 -156.[Abstract]
Morley JE. Andropause, testosterone therapy, and quality of life in aging men. Cleve Clin J Med. 2000; 67: 880 -882.[Medline]
Morley JE. Anorexia, body composition, and ageing. Curr Opin Clin Nutr Metab Care. 2001; 4: 9 -13.[Medline]
Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand binding systems. Anal Biochem. 1980;107: 220 -239.[Medline]
Papadopoulos V. Peripheral-type benzodiazepine/diazepam bind
inhibitor receptor: biological role in steroidogenic cell function.
Endocr Rev.
1993; 14:
222
-240.
Papadopoulos V, Amri H, Boujrad N, et al. Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids. 1997a; 62: 21 -28.[Medline]
Papadopoulos V, Amri H, Li H, Boujrad N, Vidic B, Garnier M.
Targeted disruption of the peripheral-type benzodiazepine receptor gene
inhibits steroidogenesis in the R2C Leydig tumor cell line. J Biol
Chem. 1997b;272:
32129
-32135.
Papadopoulos V, Mukhin AG, Costa E, Krueger KE. The peripheral-type
benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis.
J Biol Chem.
1990; 265:
3772
-3779.
Potts JO, Creange JE, Harding HR, Schane HP. Trilostane, an orally active inhibitor of steroid biosynthesis. Steroids. 1978; 32: 257 -267.[Medline]
Privalle CT, Crivello J, Jefcoate CR. Regulation of
intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P450
in rat adrenal gland. Proc Natl Acad Sci USA.
1983; 80:
702
-706.
Stocco DM, Clark BJ. Regulation of the acute production of steroids
in steroidogenic cells. Endocr Rev.
1996; 17:
221
-244.
Van den Beld AW, de Jong FH, Grobbee DE, Pols HA, Lamberts SW.
Measures of bioavailable serum testosterone and estradiol and their
relationships with muscle strength, bone density, and body composition in
elderly men. J Clin Endocrinol Metab.
2000; 85:
3276
-3282.
Vermeulen A. Androgen replacement therapy in the aging male—a
critical evaluation. J Clin Endocrinol Metab.
2001; 86:
2380
-2390.
West LA, Horvat RD, Roess DA, Barisas BG, Juengel JL, Niswender GD.
Steroidogenic acute regulatory protein and peripheral-type benzodiazepine
receptor associate at the mitochondrial membrane.
Endocrinology.
2001; 142:
502
-505.
Zirkin BR, Chen H. Regulation of Leydig cell steroidogenic function
during aging. Biol Reprod.
2000; 63:
977
-981.
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