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Department of Pediatrics, University of Zurich, Division of Endocrinology/Diabetology and Clinical Chemistry/Biochemistry, 8032 Zurich, Switzerland
Address all correspondence and requests for reprints to: Dr Anna Biason-Lauber, Department of Pediatrics, University of Zurich, Division of Endocrinology/Diabetology Steinwiesstrasse 75, 8032 Zurich, Switzerland. E-mail: alauber{at}kispi.unizh.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase and 17,20-lyase, essential for the synthesis of
cortisol and sex hormone precursors, respectively. The two activities
are differentially regulated in a tissue and developmental
stage-dependent fashion. Leptin might play a role in such differential
control. Low dose leptin caused a significant increase in 17,20-lyase
activity in adrenal NCI-H295R cells expressing leptin (OB) receptor
(OB-R), without significant sustained influence on the
17-hydroxylase activity. To analyze the time dependence of this
leptin effect, the impact of long and short-term leptin treatment was
studied. To assess the relationship with the OB-R signal transduction
pathway, the same experiments were performed in intact cells and in a
reconstituted system. The long- and short-term studies in intact cells
and in microsomes suggest that the 17-hydroxylase activity of CYP17
can be promptly stimulated by leptin, but that the effect is transient.
In contrast, physiological doses of leptin steadily enhance 17,20-lyase
activity. This influence is direct, OB-R specific and dependent on the
integrity of the signal transduction pathway. The 17,20-lyase activity
stimulation relies on phosphate incorporation, as demonstrated by the
loss of leptin-dependent 17,20-lyase stimulation after phosphate
removal, and by the fact that the DHEA production appears
to be related exclusively to the presence of phosphorylated CYP17,
independently from novel protein synthesis. The mechanism underlying
the observed events seems to involve CYP17 phosphorylation, a feature
of the OBR signal transduction pathway, and a process already shown to
be crucial for 17,20-lyase activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among several possible factors, leptin deserves particular notice. Leptin, the product of the ob gene, is an adipocyte-derived peptide hormone that exerts major effects on energy homeostasis. In addition, it affects reproductive function in animals (1, 2) and humans. Patients lacking leptin or its receptor show no signs of sexual maturation at the expected time of puberty (3, 4). In normal boys and girls, leptin increases some time before the gonadotropin peak, and its variations appears to be independent from changes in auxological parameters, mainly in boys. (5). These data suggest a permissive action of leptin for the initiation of sexual development. Interestingly, the rise in leptin levels occur at age 8–10, roughly corresponding to the time of adrenarche.
The clinical and experimental data on the relationship between leptin and adrenal maturation at the time of adrenarche are controversial. Although some authors failed to observe a relationship between DHEAS levels and leptin during pubertal development in normal boys (6), others reported a positive correlation between DHEA and leptin in patients with the Prader-Labhart-Willi syndrome, independently from body mass index (BMI). (7). Recent experimental studies demonstrated a direct inhibition of cortisol release by leptin in bovine (8), human, and rat (9) adrenal cells in culture, although at supraphysiological doses (100–1000 ng/ml, normal adult plasma levels 16.9 ± 10.9). The mechanism underlying such reduction seems to be a decrease in CYP17 messenger RNA (mRNA) accumulation. No data are available concerning a direct effect of leptin on CYP17 enzymatic activities. We therefore decided to investigate this aspect of CYP17 activity regulation in NCI-H295R adrenocortical carcinoma cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Human adrenocortical carcinoma cells NCI-H295R (ATCC No.
CRL-2128) and human ovarian adenocarcinoma cells NIH OvCar3 (ATCC No.
HTB-161) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured as
recommended by the purchaser. For the initial plating, 7.5 x
104 cells were seeded on 35-mm plates. Medium was
replaced every 24 h. Experiments were performed 48 h
thereafter (confluence about 70%). Leptin treatment was then initiated
at the given concentrations and discontinued at the given time points.
Because most cells detached from the culture plate under leptin
treatment, the medium was collected, and the detached cells were
harvested by centrifugation and replated. The cells that remained
adherent were trypsinized and collected by centrifugation. The
viability of the detached cells was controlled by trypan blue
exclusion.
For all the following experiments, the cells expressing OB-R (see later) were selected by treatment with 30 pM leptin for 24 h, collected by centrifugation, and replated in the absence of leptin. These cells were normally reattached after 12 h. Upon further treatment with leptin, the selected OBR+ cells detached again, and the following experiments were conducted in suspension. To assay enzyme activity and phosphorylation state of CYP17, such selected cells were used, unless otherwise indicated. As further characterization of the populations of NCI-H295R cells, and to check the specificity of leptin effect on CYP17, the classical stimulator 8Br-cAMP (200 µM) was added to the cells either 2 h prior or simultaneously with the addition of steroid precursors. The expected products (17OH-progesterone and DHEA) were then measured in cell extracts (see later).
For the experiments where the progesterone production was investigated, the cells were plated at a concentration of 50 x 103 cells/well in 96-well plates and cultivated for 24 h in medium plus serum, and consequently selected for OBR using leptin for 6 h. The stimulation was performed using 200 µM 8Br-cAMP, 30 pM leptin, and 50 µM cholesterol or 22R-HC, for 30', 6 and 2 h, respectively.
Protein synthesis inhibition
OBR + and OBR- cells were separated by preincubation with 30
pM leptin for 6 h. Two hours before the addition of
the protein synthesis inhibitor cycloheximide (Sigma),
steroidogenic precursors were added as described below. At time 0
min, 40 µM cycloheximide and 30 pM
leptin were added to NCI-H295R cells with or without 0.1 mCi/ml
35S-methionine or 32P
orthophosphate (32Pi) (200 µCi/ml). The
reaction was stopped by removing the medium and lysing the cells at
times 2 h 30 min, 4 h 30 min and 6 h 30 min. Enzyme
activity and immunoprecipitable CYP17 labeled protein content were
analyzed as described below. Western blot analysis was performed using
standard procedures.
RNA analysis: RT-PCR and Northern blot
Total RNA was extracted from detached and attached cells in the
absence or presence of 30 and 100 pM leptin (24 h
treatment) using the RNeasy minikit (QIAGEN, Hilden,
Germany). RT was performed using superscript reverse
transcriptase (Life Technologies, Inc., Gaithersburg,
MD). PCR amplification of OBR exons 15–17 was performed as
described (4). PCR products were separated by electrophoresis on 1%
agarose gel and Southern blot was performed following standard
procedures. A -32P-dATP labeled human OB-R
complementary (cDNA) was used as hybridization probe. PCR amplification
of human StAR, CYP11A1, and adrenodoxin (ADX) was done using the
following primers and cycling conditions: StAR sense
5'-GCAGCAGCAGCGGCGGCAGCAG-3' (designed to distinguish between the
active gene and the pseudogene); antisense 5'-ATGAGCGTGTGTACCAGTGCAG
3'; 94 C for 45 sec, 64 C for 30 sec, 72 C for 60 sec x 30
cycles; CYP11A1 sense 5'-TACCAGAGACCCATAGGAG-3', antisense
5'-CGCACACCCCAGCCAAA-GC-3'; 94 C for 45 sec 58 C for 45, 72 C for
60 sec x 30 cycles; ADX sense 5'-CTGCCGCCCCCGCCTCTT-3', antisense
5'-CAGTCTGAACACATAGCTT-3', 94 C for 45 sec, 58 C for 45 sec, 72 C for
60 sec x 30 cycles. For human cytochrome b5
amplification the primers and PCR conditions were as follows: sense
5'-TGGCAGAGCAGTCGGACG-3', antisense 5'-GCTCTTCCTGCGCTGACTTCTG-3', 94 C
for 45 sec, 52 C for 60 sec, 72 C for 60 sec x 40 cycles.
Northern blot analysis was performed using standard procedures,
hybridizing 20 µg of total RNA extracted from OBR+ NCI-H295R cells to
a radiolabeled human CYB5 cDNA fragment (bp 1–460).
In vivo metabolic labeling and immunoprecipitation
Cells were metabolically labeled with either
32P orthophosphate (32Pi)
(200 µCi/ml, NEN Life Science Products, Boston,
MA) for1 h in phosphate-free medium (Life Technologies, Inc. 11963–022) or with 35S-methionine
(100 µCi/ml, NEN Life Science Products, easytag) for
2 h in methionine-free medium (Life Technologies, Inc.). Labeled cells were lysed in 1 x PBS, 1.5
mM MgCl2, 1 mM EDTA, 1% Triton-X, 10%
glycerol in the presence of protease inhibitors (phenylmethanesulfonyl
fluoride 34 µg/ml, 0.7 µg/ml pepstatin, 5 µg/ml leupeptin,
Roche Molecular Biochemicals) and phosphatase inhibitors
for the phosphorylation experiments (100 mM
sodium-fluoride, 10 mM sodium-pyrophosphate, 2
mM sodium-orthovanadate, Sigma). The lysates
were clarified by centrifugation at 15,000 x g for 10
min. The supernatants were then bound to Protein A Sepharose
(Amersham Pharmacia Biotech, Dubendorf,
Switzerland) preincubated (at least 20 min at RT) with antihuman
CYP17 antibodies (a generous gift from Prof. M. Waterman, Nashville,
TN) at a dilution 1:50.000. The cell lysates were incubated on the
Protein A Sepharose/Ab complex O/N at 4 C. The immune complexes were
then extensively washed and analyzed on 10% SDS-PAGE. Quantification
of 32P and 35S
incorporation was conducted by cutting the bands and counting. The
standardization was done by dividing the 32P cpm
and 35S cpm per mg total protein. The experiments
were repeated three times.
Enzyme assay
Intact cells. Steroidogenic precursors (progesterone for
17-hydroxylase activity and 17OH pregnenolone for 17,20-lyase
activity) were added at the concentration of 750 ng/ml after suspension
in 1 x phosphate buffer. Six hours after addition, supernatant
were removed for the time point 0 and leptin treatment was initiated.
The same procedure was adopted for the several time points. All samples
were kept frozen at -20 C until measured. To assay the intracellular
concentration of steroids, cell lysates were prepared as described
above, protein content was measured using Bio-Rad Laboratories, Inc. protein assay reagents, the steroids were extracted with
ethyl acetate/isooctane (1:1) and assayed by RIA.
Microsomes (reconstituted system). Microsomal membranes were prepared from OBR + NCI-H295R cells, as described (10). The total protein content was 1.2 mg/ml. CYP17 activity was measured by incubating 10 µg of microsomal protein with 500 nmol of progesterone or 17OH-pregnenolone for 30 min, as previously described (10).
Long term. Thirty picomoles leptin were added at time 0 min, and incubations were discontinued at time 2, 4, 6, 10, 12, and 24 h thereafter, when cell extracts were prepared, steroid extracted and assayed in duplicate.
Short term. The experimental procedure is the same as described for the long-term experiments, with the exception of the time points: every 5 min over 1 h.
The alkaline phosphatase experiments were performed after 60-min leptin treatment (30 pM) in intact cells, subsequently lysed as previously described. The cell lysates were preincubated in ice-cold 50 µM Tris·Hcl, pH 8/1 mM, MgCl2 at 37 C for 10 min. One unit alkaline phosphatase (Roche Molecular Biochemicals) (or an equivalent volume of water as control), was added and the reaction was stopped at time 0, 2, 4, 8, 12, 16, and 20 min with 50 mM EDTA. To assess more precisely the role of phosphate, 2 mM of the phosphatase inhibitor sodium-orthovanadate was added to the lysis buffer at each time point. The enzyme activity was measured at the several time points as described above.
The products were extracted with ethyl acetate/isooctane (1:1),
concentrated by evaporation, resuspended in 500 µl RIA buffer (20
mM KH2PO4; 47
mM
Na2HPO4.2H2O;
5 mM NaN3;1 g/liter bovine
-globulin) and assayed by RIA.
Progesterone, 17OH-progesterone, and DHEA were measured in duplicates by RIA using Diagnostic Products Corp. kits (Los Angeles, CA).
Statistical analysis
All values, representing the results of three independent
experiments, are expressed as mean ± SD and subjected
to t test analysis (paired).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). That provided us with a
selection assay for leptin responsive cells. The cells remaining
adherent, expressing CYP17 but not expressing OB-R, were used as
negative control. There was no significant qualitative time-dependent
change in OB-R expression upon leptin addition in detached NCI-H295R
cells (Fig. 1, C and D). Normalization of the RT-PCR data to expression
of GAPDH gene (not shown) demonstrated that the differences in cDNA
amount between the long- and the short-term treatments are due to
variations in initial RNA content. Leptin treatment (30 pM
for 24 h) caused a significant increase in 17,20-lyase in the
OB-R+ NCI-H295R cells, but not on the OB-R- cells, and had no
significant influence on the 17-hydroxylase activity in both OB-R +
or OB-R- cells (Fig. 2). Although the
actual steroid product values differ between the secreted (medium) and
the intracellular milieu, the general tendency appears to be the same,
suggesting that there is an equilibrium between intra and
extra-cellular steroids secretion, and that the increase of
DHEA is not due to an alteration of the membrane
permeability.
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Additional evidence that the stimulatory effect of leptin on CYP17
enzymatic activity is specific and the OBR- cells are actually capable
to respond to stimuli, is given by the demonstration that the classical
stimulator of CYP17, cAMP, is able to significantly enhance
17-hydroxylase and 17,20-lyase activities in both OBR+ and OBR-
cells : 17OH-progesterone goes from 100 ± 5 to 403 ± 10
in OBR + and from 110 ± 10 to 390 ±7 ng/mg protein in OBR
–cells; DHEA in OBR + cells: basal 75 ± 4 ng/mg
protein, stimulated 350 ± 7 ng/mg protein; in OBR-cells: basal
68 ± 6 ng/mg protein vs. stimulated 345 ± 12
ng/mg protein. All the differences are significant: P
< 0.0001. The time of addition (-2 h or time 0) had no significant
influence on the outcome.
To analyze the time dependence of leptin effect, a long- and short-term leptin treatment was carried out. To assess the involvement of the OB-R signal transduction pathway in the observed events, the long- and short-term experiments were performed in intact cells and in a microsomal protein preparations in a reconstituted system.
17-hydroxylase activity
Intact cells. As shown in Fig. 3, A and B, although in living cells an
apparent effect of leptin on 17OH-progesterone production was
detectable after 4 h, this effect disappeared in the following
time points. Microsomes (OBR+): Upon short-term leptin
treatment, 17OH-progesterone production showed a biphasic stimulation
pattern, with a rapid initial increase (25–30 min) followed by a
plateau and a steeper raise in the later time points. No elevation of
17OH-progesterone was seen in the absence of leptin (Fig 3C). Longer
treatment appears to have an inhibitory effect on 17-hydroxylase
activity, although with an alternating pattern (Fig. 3D). The same
trend was seen in the absence of leptin (Fig. 3D), suggesting an
unspecific mechanism maybe related to cellular cyclic processes. The
long- and short-term studies in intact cells and in reconstituted
system thus suggest that the 17-hydroxylase activity of CYP17 can be
promptly stimulated by leptin, but that the effect is of short duration
because already 10 h after initiation of treatment, the
17-hydroxylase activity had returned to baseline (3B). This
stimulation appears to be dependent on the presence of leptin in
microsomes of OBR+ cells (Fig. 3, C and D), and/or of its receptor in
intact cells, only when the treatment is kept long enough (2 h) (Fig. 3B). On the other hand, the 17-hydroxylase activity seemed to be
enhanced independently from this signal transduction pathway (Fig. 3, C
and D).
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suggesting an OB-R specific effect. In contrast to 17OH-progesterone,
the DHEA synthesis increased progressively between 6 and
12 h after initiation of treatment, and stayed constant up to
24 h (Fig. 4B). This event appears to be OB-R dependent because
the OB-R negative cells did not show any difference in CYP17 enzymatic
activity upon leptin addition. Microsomes (OBR+): When the
same experiment was carried out in the reconstituted system, no such
differential effect was seen, suggesting that the cell-free system does
contain the elements necessary to stimulate 17,20-lyase activity (Fig. 4C).
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, no positive correlation between CYB5 mRNA abundance and 17,20-lyase
activity was detectable.
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, A
and C). The differences in phosphate incorporation are not due to
variations in protein synthesis, as demonstrated by the comparable
amount of 35S-methionine labeled CYP17 protein (Fig. 6B). The
divergence in immunoprecipitable phosphorylated CYP17 protein between
OBR+ and OBR- cells, remains significant when the amount of
32P radioactivity is corrected by
35S-dependent radioactivity. The same is true for
the nonsignificant differences in phosphate incorporation under leptin
treatment in both cell populations (Fig. 6C). This higher phosphate
content in the CYP17 protein of leptin responsive cells correlates with
17,20-lyase activity, that appears to be higher in these cells already
at the first treatment time points (Figs. 3
and 4). Although NIH-OvCar3
cells express CYP17, no phosphorylation of the protein was seen in
these cells (not shown). The elimination of the O-phosphomonoesters
from the hydroxyamino acids (ser/thr and tyr) by way of alkaline
phosphatase treatment, selectively inhibited the DHEA
formation in OBR expressing cells (Fig. 7A), without influence on the
17-hydroxylase activity (Fig. 7B), confirming the dependence of
17,20-lyase activity on phosphate incorporation. The specificity of the
effect was confirmed by the abolition of the differences in CYP17
activities upon inhibition of the phosphate removal by
sodium-orthovanadate treatment (Fig. 7). The reliance of 17,20-lyase
activity stimulation on posttranslational modification is confirmed by
the fact that complete inhibition of novel protein synthesis
(cycloheximide treatment, 8B), did not significantly alter the
DHEA increase under leptin treatment in OBR+ cells (Fig. 8A) at early time points (2.5 h). The
later decrease in DHEA production correlates with a
reduction in CYP17 phosphorylated protein down to 25% of baseline
(Fig. 8C) and not to decreased protein content, as demonstrated by the
Western blot analysis (Fig. 8D). Whether this decline in phosphate
incorporation depends on an influence of cycloheximide on the cellular
phosphorylation/dephosphorylation equilibrium remains to be
established. 17-hydroxylase activity is not surprisingly independent
from phosphate incorporation (Fig. 8C).
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, the addition
of leptin does not influence conversion of 22R-HC to progesterone in
OBR + NCI-H295R cells alone or in combination with cAMP. The addition
of cholesterol to the culture medium caused an inhibition of
progesterone production in cAMP stimulated cells, but not in basal
conditions. At present time, we have no explanation for this
phenomenon.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase and
17,20-lyase. The dual function of this enzyme allows direction of the
steroid precursors along several pathways: 1) 17-hydroxylated
products with the intact side chain are precursors of cortisol, as in
adrenal zona fasciculata; 2) generation of C19 steroids
by both, 17-hydroxylation and 17,20-cleavage, directs substrates
toward the formation of sex hormones, as in adrenal zona
reticularis and in the gonads; 3) in the adrenal zona
glomerulosa, which lacks the CYP17 activities, pregnenolone is
converted to mineralocorticoids. The two activities of CYP17 are differentially regulated in a tissue- and time-dependent fashion. The redox partner abundance and posttranslation modifications, such as phosphorylation, have been demonstrated to play an essential role in the activation of 17,20-lyase activity (10, 11, 12, 13, 14). Nevertheless, the physiological factor(s) involved in such activation are still unknown. Clarification of the control mechanisms governing the two activities is an essential step in the understanding of adrenarche and the onset of puberty.
Among the several possible agents, leptin has recently gained importance. It acts through the leptin receptor OB-R, a single-transmembrane-domain receptor of the class I cytokine receptor family (15). These receptors are known to act through JAK and STAT proteins. Typically, JAK proteins are constitutively associated with membrane-proximal sequences of the receptor intracellular domain (ICD) and phosphorylate tyrosine residues of the receptor ICD upon ligand binding. The phosphorylated ICD then provides a binding site for a STAT protein, which is then activated. The activated STAT protein then translocates to the nucleus and stimulates transcription. In the case of OB-R, JAK 2 and STAT3 are involved in the signal transduction and transcriptional activation. Because the OB-R signal transduction pathway operates through phosphorylation of JAK and STAT proteins, the mechanism underlying the observed events most likely involves phosphorylation, a process already shown to be crucial for 17,20-lyase activity (10).
Leptin can exert its action on multiple targets. In fact, although it appears that posttranslation modifications are crucial for 17,20-lyase activity, the role of substrate accessibility in the increased DHEA production must be assessed. Because the precursor for all steroids is cholesterol, leptin might influence cholesterol availability by affecting either its transfer to the inner mitochondrial membrane or the rate-limiting step of steroidogenesis, i.e. the cleavage of the side chain of cholesterol to yield pregnenolone. Possible targets of such action would therefore be steroidogenic acute regulatory protein (StAR) or cytochrome P450 side chain cleavage (CYP11A1), respectively. The mechanism regulating this putative influence likely relies on phosphorylation. In fact, although phosphorylation might not be required for StAR mitochondrial import, phosphorylation of StAR is directly linked to steroidogenic response of the cell to hormone stimulation. (18). The fact that the enzymatic system is below saturation conditions, grants the bases for this hypothesis. We therefore performed experiments to assess the possibility that the rise in DHEA production under leptin treatment is due to stimulation of StAR and/or CYP11A1 activities, or inhibition of conversion of DHEA to androstenedione, catalyzed by 3ß-hydroxysteroid dehydrogenase. The lack of influence of leptin on progesterone production suggests that this is not the case, and reinforces the thesis of a direct effect of leptin on CYP17 catalytic potential.
Another possible mechanism by which leptin stimulates 17,20-lyase activity may reside in an increase of the availability of cytochrome b5, the alternative electron donor for CYP17. The absence of a positive correlation between CYB5 gene expression and 17,20-lyase activity implies that leptin-stimulated DHEA production is not primarily attributable to an increase of b5:CYP17 ratio.
The effect of leptin on 17-hydroxylase activity seems to be
consistently independent from cell integrity, suggesting that the
underlying mechanisms are different from the OBR second messenger
pathway. Nevertheless, this effect is dependent on the presence of
leptin and its receptor. Further studies are necessary to clarify
whether the observed phenomenon represents a specific event or an
experimental artifact.
On the other hand, our data suggest for the first time a direct role of leptin already at physiological concentrations in the regulation of 17,20-lyase activity. This influence is direct, OB-R specific and depends on the integrity of the signal transduction pathway. The effect of leptin treatment can be seen at early stages, implicating a role of leptin in the acute regulation of adrenal androgen synthesis, probably associated with posttranslational modifications. The dependence of 17,20-lyase activity on the phosphorylation state is confirmed by two lines of evidence: first, the differences in phosphorylation state of CYP17 in OBR+ and OBR- cells correlate to the differences in DHEA production in these two cell populations. The precise mechanism responsible for these observation remains to be elucidated. Second, the loss of 17,20-lyase activity after O-phosphomonoesters removal by alkaline phosphatase. The fact that inhibition of the dephosphorylation (sodium-orthovanadate treatment) abolishes the phenomenon further corroborates the hypothesis. Moreover, the effect of leptin treatment on 17,20-lyase activity is not dependent upon new protein synthesis because complete block of translation does not change the increment in DHEA production. Such DHEA synthesis, therefore, appears to rely on the presence of phosphorylated CYP17 protein. The lack of differences in immunoprecipitable phosphorylated CYP17 protein under leptin treatment, suggests the need of more specific assays for the detection of single phosphorylated residues in the CYP17 protein. Nevertheless, a correlation between the basal phosphorylation state of CYP17 protein and basal 17,20-lyase enzymatic activity appear to exist, as demonstrated by the differences in DHEA production between OBR+ and OBR- NCI-H295R cells. It remains to be established whether phosphorylation must take place directly on CYP17 or engages some other intermediary protein that, once activated, in turn, promotes phosphorylation of CYP17.
Our results contrast with those of others (16) reporting a
leptin-dependent down-regulation of CYP17 mRNA with consequent decrease
of steroid production in human adrenal cells. Several points can be
made to explain this discrepancy. First, the leptin doses used in our
experiment, resembling closer the physiological concentrations, are
much lower than those used by Glasgow et al. Second,
incubation time appears to play a role, at least with regard to the
17-hydroxylase activity. Third, it appears that the reported
inhibitory effect of leptin is not direct because no data are presented
showing the influence of leptin alone, i.e. without ACTH on
CYP17 mRNA or activity. Although ACTH is essential for the regulation
of cortisol production in the zona fasciculata, it appears
to play a more marginal role in governing the 17,20-lyase activity in
the zona reticularis, as demonstrated by the lack of
increase of ACTH levels at adrenarche (17). Furthermore, the described
cases of either leptin deficiency (3) or leptin resistance (4) show no
abnormal ACTH or cortisol levels, suggesting the absence of gross
defects in the hypothalamic-pituitary adrenal axis and implicate a more
marginal role of leptin in the physiological regulation of
17-hydroxylase activity. On the other hand, the lack of production
of any sex hormones, mirrored by the failure of sexual development in
these patients, confirms the physiological importance of leptin in the
stimulation of the 17,20-lyase activity of CYP17.
In conclusion, we demonstrated that leptin in physiological amounts is
important in the differential regulation of 17,20-lyase vs.
17-hydroxylase activity of CYP17 enzyme. The action of leptin
appears to imply the control of CYP17 phosphorylation, it requires the
presence of the leptin receptor and the integrity of the OB-R signal
transduction pathway, without the need of concomitant activation of
other signal transduction routes, such as that coupled to the MC2-
receptor (ACTH receptor). Our data implicate leptin in the acute,
although not immediate, and long-term stimulation of 17,20-lyase
activity in human adrenal cells and represent a step forward in the
understanding of the mechanisms governing adrenarche.
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Acknowledgments |
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Footnotes |
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Received August 4, 1999.
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E. Charmandari, M. Weise, S. R. Bornstein, G. Eisenhofer, M. F. Keil, G. P. Chrousos, and D. P. Merke Children with Classic Congenital Adrenal Hyperplasia Have Elevated Serum Leptin Concentrations and Insulin Resistance: Potential Clinical Implications J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2114 - 2120. [Abstract] [Full Text] [PDF]
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M. R. Palmert, D. L. Hayden, M. J. Mansfield, J. F. Crigler Jr., W. F. Crowley Jr., D. W. Chandler, and P. A. Boepple The Longitudinal Study of Adrenal Maturation during Gonadal Suppression: Evidence That Adrenarche Is a Gradual Process J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4536 - 4542. [Abstract] [Full Text] [PDF]
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N. Cherradi, A. M. Capponi, R. C. Gaillard, and F. P. Pralong Decreased Expression of Steroidogenic Acute Regulatory Protein: A Novel Mechanism Participating in the Leptin-Induced Inhibition of Glucocorticoid Biosynthesis Endocrinology, August 1, 2001; 142(8): 3302 - 3308. [Abstract] [Full Text] [PDF]
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, 0 pM; 
, 30 pM; 
, 100
pM.
) by 6 or 24 h
leptin treatment (30 pM). OBR+ cells were preincubated with
750 ng/ml steroidogenic precursors at time -6 h for the intact
cells experiments and -30 min for the assay in
microsomes, in the presence (•) or absence (
) of 30
pM leptin. In more details, 30 pM leptin were
added at time 0 and incubations were discontinued at time 2, 4, 6, 10,
12, and 24 h, thereafter, when cell extracts were prepared,
steroid extracted and assayed in duplicate. All values are expressed as
mean ± SD and represent the result of three
independent experiments. The data were subjected to t
test analysis (paired): ns, nonsignificant; *, P <
0.025; ***, P < 0.005. Note the differences in
x-axis scale.
). The effect is specific because OBR- cells
(
). The data were subjected to t test
analysis (paired): **, P < 0.01; ***,
P < 0.005.
