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Cattedra di Andrologia (M.C., A.M.I., A.F.), Universita’ La Sapienza, Policlinico Umberto I, 00161, Rome, Italy; Laboratory of Neurophysiology (A.R.C.), National Institutes of Mental Health, National Institutes of Health, Bethesda, Maryland 20892; Cattedra di Endocrinologia (C.M.), Universita’ Tor Vergata, 00100 Rome, Italy; Endocrinology and Reproduction Research Branch (M.L.D.), National Institutes of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892; and the Department of Endocrinology (A.F.), St. Bartholomew’s Hospital, EC1A 7BE London, United Kingdom
Address all correspondence and requests for reprints to: Andrea Fabbri, M.D., Ph.D., Department of Endocrinology, St. Bartholomew’s Hospital, West Smithfield, EC1A 7BE, London, United Kingdom. E-mail: a.fabbri{at}caspur.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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60% reduction,
IC50 = 20 ng/ml) but no change in basal
androgen release. Also, leptin (150 ng/ml) amplified hCG-induced
intracellular cAMP formation (1- to 2-fold) without modifying basal
cAMP levels. Subsequent experiments showed that leptin inhibited
8Br-cAMP-stimulated T production, indicating that leptin’s effect is
exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T
secretion was accompanied by a significant reduction of androstenedione
and a concomitant rise of the precursor metabolites pregnenolone,
progesterone, and 17-OH-progesterone, conceivable with a leptin-induced
lesion of 17,20 lyase activity. Separate experiments performed with the
MLTC-1 cells (not expressing cytochrome P450–17
) showed that
leptin, though amplifying hCG-stimulated cAMP production, did
not modify hCG-stimulated pregnenolone and progesterone release. These
results further indicate that leptin action on steroidogenesis occurs
downstream of progesterone synthesis. Northern Blot experiments showed
no acute effect of leptin on cytochrome P450–17 messenger RNA
accumulation in rat Leydig cells in basal and hCG-stimulated
conditions, excluding that the rapid changes observed were caused by
messenger RNA degradation. In conclusion, these findings, for the first
time, show that leptin has direct, receptor-mediated actions on rodent
Leydig cells in culture, at concentrations within the range of obese
men. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The long and short isoforms of leptin receptor are widely
expressed in peripheral tissues (14, 15, 16), and recent reports suggest
that leptin could have important peripheral actions, including effects
on the endocrine pancreas (17, 18), the hematopoietic stem cells (19, 20), and steroidogenic tissues such as the ovary (21, 22, 23, 24) and the
adrenals (25, 26, 27). In particular, leptin has been found to suppress
insulin-induced progesterone and 17ß-estradiol production by isolated
bovine granulosa cells (21), prevent insulin-induced progesterone and
androstenedione secretion in bovine ovarian thecal cells (22), and
impair hormonally-stimulated release of 17ß-estradiol by rat
granulosa cells in culture (23). Furthermore, leptin has been shown to
inhibit glucocorticoid secretion by cultured bovine adrenocortical
cells (25) and to significantly reduce the ACTH-stimulated cortisol
release from normal human and rat adrenal gland (26), as well as the
ACTH-stimulated expression of cytochrome P450–17 messenger RNA
(mRNA) accumulation in human and bovine adrenal cells in culture (25, 27).
These data suggest that leptin may have relevant peripheral actions on target steroidogenic cells. In the present study, we have tested the hypothesis that, in addition to its central nervous system effects on the reproductive axis, leptin may exert direct effects in the control of steroid production from isolated rodent Leydig cells in culture. To test this hypothesis, we first studied leptin receptor expression in isolated adult rat Leydig cells and in a murine Leydig tumor cell line (MLTC-1), then we investigated the effects of leptin on basal and human (h)CG-stimulated steroid production by rat Leydig cells and MLTC-1 cells in culture.
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Materials and Methods |
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MLTC-1 cells propagation and culture
Stock cultures of MLTC-1 cells were grown and maintained in
RPMI-1640 medium (Whittaker M.A. Bioproduct) containing 25
mM HEPES, 300 mg L-glutamine/liter, 2000 mg
glucose/liter, and supplemented with 10% FCS and 50 mg/liter
gentamicin. The cells were subcultured by trypsinization [0.05%
(wt/vol) trypsin], and experimental cultures were plated in 6-well
tissue culture dishes and grown to 70–80% confluency before
initiation of the experiments. The medium was then removed, and the
cells were washed three times with PBS and returned to fresh RPMI
medium supplemented with 0.1% BSA, 50 mg/liter gentamicin, and 0.125
mM 3-methylisobutyl xanthine. Cell viability was determined
by the trypan blue dye-exclusion test (30).
Incubation and assays
The purified rat Leydig cells and MLTC-1 cells were plated in
6-well tissue culture dishes (1.5 x 106
cells/well·2 ml) and then incubated at 37 C under 95%
[O2] and 5% [CO2] for the designed time
period (see figure legends), in the presence or absence of saturating
doses of hCG (1 ng/ml for rat Leydig cells, 20 ng/ml for MLTC-1 cells;
CR 121, preparation kindly provided by the Center for Population
Research, National Institutes of Child Health and Development,
Bethesda, MD), or 8Br-cAMP (final concentration 1 mM)
(Sigma Chemical Co., St. Louis, MO) with or without
recombinant murine leptin (R&D Systems, Minneapolis, MN) at different
concentrations, for the designed time period (see figure legends). At
the end of incubation, media were removed and centrifuged at 250
g for 12 min, and the supernatants were saved for the assay
of extracellular pregnenolone, progesterone, (from both rat Leydig
cells and MLTC-1 cells), 17-OH-progesterone, androstenedione, and
testosterone (T) (only from rat Leydig cells). All plated cells were
also processed for the analysis of intracellular cAMP, as described
elsewhere (31). The measurements of steroids and cAMP were performed by
RIA, as previously described (32).
All the experiments were performed at least three times, in triplicate; results are the mean ± SE unless otherwise specified. Statistical significance was determined by ANOVA test.
Cytochrome P450–17 and c-fos expression
In other experiments, purified rat Leydig cells were plated in
100 x 20-mm tissue-culture dishes (7.5 x 106
cells/dish·10 ml) and incubated in the same conditions as above, for
30 min, in the presence or absence of hCG (1 ng/ml), with or without
recombinant murine leptin (150 ng/ml). Cells were then washed three
times with ice-cold PBS, scraped, and centrifuged at 250 x
g for 12 min. Total RNA was isolated from cell pellets by
using TRIzol RNA reagent (Life Technologies, Inc.,
Gaithersburg, MD) followed by deoxyribonuclease I (Life Technologies, Inc.) treatment to remove DNA contamination. The
total RNA samples (15 µg for each lane) were resolved in 1.5%
agarose gel containing 10 x
3-[N-morpholino]propanesulfonic acid and
formaldehyde, transferred onto a Gene screen membrane (Biotechnology
System, NEN Life Science Products, Boston, MA). The fixed
membrane was prehybridized with a buffer containing 6 x SSC,
5 x Denhardt’s and 0.5% SDS (Quality Biological
Inc., Gaithersburg, MD) and hybridized overnight with a
32P-labeled rat P450–17 complementary DNA (cDNA) probe
(33). Thereafter, the membrane was washed twice in 2 x SSC-0.1%
SDS at 65 C for 15 min each and twice in 0.5 x SSC-0.1% SDS for
10 min each. Hybridization was evaluated by autoradiography. The same
membrane was stripped by two washes of 10 min each in 1% SDS at 85 C
and one wash of 2 min in 0.2 x SSC. After confirmation of the
removal of radioactivity, by exposing the membrane to x-ray film
overnight, the membrane was hybridized again with a
32P-labeled rat c-fos cDNA probe, as described
elsewhere (34), and hybridization was evaluated by autoradiography.
Quantification of P450–17 and c-fos mRNA was evaluated
by Phosphorimage Scanner (Molecular Dynamics, Inc.,
Sunnyvale, CA) after normalization with ß-actin, as previously
reported (33).
Leptin receptor expression
Total RNA from primary rat Leydig cells, MLTC-1 cells, and
rat and mouse, full-brain (positive) control was obtained as previously
described. RNA was then subjected to RT-PCR using a kit from
Perkin-Elmer Corp. Europe B. V. (Cretkrevz,
Switzerland) (Gene Amp RNA PCR kit). First-strand cDNA was synthesized
from 1 µg of total RNA using oligo dT primers and an annealing
temperature of 65 C, and then amplified by PCR in 50 µl of reaction.
Four sets of primers, based on rat and mouse leptin receptor sequences,
were chosen (Fig. 1
). Set 1 primers
[5'-ATGCTGTGCAGTCACTCAGTG-3' (sense, nucleotides 2284–2303) and
5'-CAACTCCTTCCATAAATACTGGG-3' (antisense, nucleotides 2526–2503), rat
sequence, GenBank accession no. U52966;
5'-GTGCTGTGGAGTCACTCAGTG-3' (sense, nucleotides 2210–2230) and
5'-CAACTCCTTCCATAAATACTGGG-3' (antisense, nucleotides 2452–2430),
murine sequence, GenBank accession no. U49110] generate a 242-bp PCR
product corresponding to the extracellular domain common to all Ob-R
isoforms. Set 2 primers [5'-GATATTTGGTCCTCTTCTTCTGG-3'
(sense, nucleotides 2786–2809) and 5'-AGTTGTGGTGAAATCACATTGG-3'
(antisense, nucleotides 3223–3201), rat sequence, GenBank accession
no. U52966; (5'-GATATTTGGTCCTCTTCTTCTGG-3' (sense, nucleotides
2712–2735) and 5'-AGTTGTGGTGAAATCATGGTGGG-3' (antisense, nucleotides
3149–3127), murine sequence, gene bank accession no. U49110] generate
a 437-bp PCR product, corresponding to the intracellular domain of the
receptor, specific of the Ob-Rb isoform. Set 3 primers
[5'-ATGCTGTGCAGTCACTCAGTG-3' (sense, nucleotides 2284–2303) and
5'-ACTTCAAAGAGTGTCCGCTCT-3' (antisense, nucleotides 2668–2689), rat
sequence, GenBank accession no. D84125; 5'-GTGCTGTGGAGTCACTCAGTG-3'
(sense, nucleotides 2210–2230) and 5'-AGTCATTCAAACCATTAGTTTAGG-5'
(antisense, 2805–2782), murine sequence, GenBank accession no.
U49106] were chosen to generate, respectively, a fragment of 479 (rat)
and 595 bp (mouse), specific only of the Ob-Ra isoform, as the
downstream primers corresponded to the cDNA of the intracytoplasmic
domain of the short isoform (Fig. 1). Finally, another set of primers
(set 4) was used, combining the upstream primer of set 1 and the
downstream primer of set 2, both from rat and murine OB-Rb sequence, to
generate longer PCR products (940 bp), spanning different exons in the
extra- and intracytoplasmic regions of rat and murine OB-Rb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(A and B) is the
photograph of two 1% agarose gels with ethidium bromide staining. A
shows the presence of PCR products of the expected size from rat
full-brain total RNA (positive control, lanes 1–3) and primary rat
Leydig cells total RNA (lanes 4–6). The generated PCR products
corresponded to a 242-bp cDNA fragment of the extracytoplasmic domain
of OB-R common to all isoforms (lanes 1 and 4), and 437-bp (lanes 2 and
5) and 479-bp (lanes 3 and 6) cDNA fragments specific of the
intracytoplasmic domain of OB-Rb and OB-Ra, respectively. B shows the
same experiment as A, performed on mouse full-brain total RNA (positive
controls, lanes 1–3) and on MLTC-1 cells total RNA (lanes 4–6), using
the respective sets of primers chosen from the murine OB-R sequence
(see Materials and Methods and Fig. 1
). All expected PCR
products could be detected, with the exception of the fragment specific
for OB-Ra that could not be amplified from MLTC-1 cells (B, lane 6),
suggesting that the short isoform of leptin receptor is not expressed
in MLTC-1 cells. Another set of primers (set 4) was used and generated
940-bp cDNA fragments specific of the OB-Rb, both from rat Leydig cells
(A, lane 8) and from MLTC-1 cells (B, lane 8). Lane 7 shows the
presence of the 940-bp fragment from rat and mouse full-brain total RNA
(A and B, respectively). Tentative confirmations of these data, by
Northern Blot analysis of rat Leydig cells and MLTC-1 cells mRNA, were
negative (data not shown), suggesting that, in these cells, the level
of expression of leptin receptor is not abundant.
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shows basal and hCG-stimulated (1
ng/ml) T secretion by rat cultured Leydig cells, after 30 and 90 min of
incubation with leptin (150 ng/ml). Leptin significantly inhibited
hCG-stimulated T production after 30 min (13.1 ± 0.3 ng/ml
vs. 27.3 ± 1.0 ng/ml, 50–60% reduction,
P < 0.01); such inhibitory effect was maintained, even
if at a lesser extent (35% reduction) after 90 min (56.5 ±
2.2 ng/ml vs. 87.3 ± 5.4 ng/ml, P <
0.01). Leptin alone did not modify T production at any of the examined
times. B shows basal and hCG-stimulated intracellular cAMP production
from the same experiment as A. Again, leptin did not alter
intracellular cAMP levels in the absence of hCG stimulus, whereas it
tended to increase hCG-induced cAMP production after 30 min and
significantly enhanced it after 90 min (56.2 ± 3.2 fmol/ml
vs. 37.3 ± 1.4 fmol/ml, P < 0.05).
Interestingly, in other experiments, leptin significantly reduced
8Br-cAMP-stimulated (1 mM) T production by 40–50%
(19.4 ± 1.3 ng/ml vs. 34.7 ± 2.1 ng/ml,
P < 0.01), suggesting that its action on
steroidogenesis is exerted beyond cAMP formation. Because leptin
effects on steroidogenesis were maximal after 30 min, subsequent
experiments all were performed by using this incubation time.
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10 ng/ml leptin, with
an IC50 of 20 ng/ml and maximal inhibition at 150
ng/ml, at which a 50–60% reduction was observed (Fig. 4). Leptin alone did not alter T
secretion at any of the concentrations tested (data not shown).
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). As
expected, hCG stimulated T production in a dose-dependent manner
(ED50 = 0.52 ng/ml). Leptin did not modify basal T
production but markedly reduced the steroidogenic response to
hCG with no significant change in hCG sensitivity (hCG
ED50, in the presence of leptin, = 0.51 ng/ml). These
results were suggestive of the presence of a leptin-induced lesion of
the steroidogenic pathway stimulated by hCG, with possible accumulation
of T precursors. Indeed, subsequent experiments showed that leptin
addition to cell cultures caused lower hCG-induced increments in the
production of androstenedione (259.3 ± 3.2 
percent change
from baseline, hCG + leptin, vs. 392.9 ± 26.1%, hCG
alone) and T (202.9 ± 9.3% vs. 382.2 ± 13.9%)
(P < 0.01), which were accompanied by higher
hCG-induced changes in the accumulation of the precursor metabolites
pregnenolone (336.7 ± 36.6% vs. 165.5 ± 32.1%)
(P < 0.05), progesterone (290.7 ± 6.7%
vs. 176.6 ± 7.8%), and 17-OH-progesterone (302.3
± 20.9% vs. 160.1 ± 21.2%) (P <
0.01) (Fig. 6). These results indicate
that leptin inhibition was likely caused by a leptin-induced defect in
17,20 lyase conversion of 17-OH-progesterone to androstenedione and T,
as also shown by the augmented percentage changes in precursor
(17-OH-progesterone) to product (androstenedione) molar ratios
(848 ± 12.6% vs. 405.6 ± 17.4%)
(P < 0.05).
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represents basal and
hCG-stimulated (20 ng/ml) pregnenolone (A), progesterone (B), and cAMP
(C) production by MLTC-1 cells in the presence or absence of leptin
(150 ng/ml). For these cells, a 20-times-higher concentration of hCG
was chosen because MLTC-1 cells are known to require approximately 70%
occupancy of the LH receptor for maximal steroid stimulation (35). In
MLTC-1 cells, leptin did not modify steroid output, either in the
absence or presence of hCG stimulus, at any of the examined times.
Interestingly, and in analogy to what was observed in rat Leydig cells,
leptin treatment amplified hCG-induced intracellular cAMP production by
60% (P < 0.05) at 90 min (C), indicating that also
MLTC-1 cells express a functional leptin receptor. Again, leptin alone
did not alter intracellular cAMP levels.
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), leptin
did not alter the accumulation of P450–17 mRNA from primary
cultured rat Leydig cells, both in basal (lanes 1–2) and
hCG-stimulated (lanes 3–4) conditions, excluding that the rapid
changes observed in hCG-induced steroidogenesis were caused by mRNA
degradation. The same membrane was stripped and hybridized with a
c-fos-specific probe to study whether leptin was able to
activate an early gene expression. In contrast to hCG that caused a
quick increase in c-fos mRNA accumulation by approximately
3-fold, as already reported by others (36, 37), leptin did not modify
c-fos expression in basal and hCG-stimulated conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Positional cloning of the leptin receptor gene showed that it encodes several alternate spliced forms (a–e, as well as others), all of which, except Ob-Re (soluble form), contain a single transmembrane domain (38). All isoforms share identical extracellular ligand binding domain with homology to the class I cytokine receptor family, but they differ at the C-terminus. Only Ob-Rb, which has the longest (303 amino acids) cytoplasmic domain, encodes all protein motifs capable of activating the JAK-STAT signal transduction pathway (39). In mice, Ob-Rb is expressed at high levels in the hypothalamus, the primary site where leptin is thought to be acting, with lower-level expression in brain, testes, and adipose tissue (40, 41). In general, in extra-brain tissues, expression levels of Ob-Rb account for only a small part of the total Ob-R expression, which is mostly represented by the expression of the short forms of the leptin receptor. These forms are products of the same gene; have no (OB-Re) or short (less than 50 amino acids) cytoplasmic domains, which contain only one (box 1) out of the two JAK binding domains; are apparently incapable of signaling via the JAK-STAT pathway; and play a role that remains to be defined (38). Besides Ob-Rb, probably only Ob-Ra, which is expressed ubiquitously and is abundant in the choroid plexus, may play important physiological roles, including leptin uptake and/or efflux from the cerebrospinal fluid, and possible increase of the local leptin concentration in a given target tissue, thereby presenting the ligand to the signaling form of the leptin receptor (41). Recent reports have shown that the Ob-Ra has distinct signal capacities and is able to transmit signals through the MAP kinase pathway in transiently transfected cells (42) and in CHO cell lines stably expressing Ob-Ra (43); the significance of these activities for leptin biology in vivo is not known. The Ob-R has been found at all points along the HPG axis, and several groups have documented evidence of Ob-R mRNA in the gonads of mice, rats, and humans (15, 16, 24). In particular, in adult mice, in situ hybridization studies performed with [35S]-labeled antisense riboprobes to the common extracellular domain of the leptin receptor have shown that Ob-R mRNA is present in the stratified epithelium of spermatic cells and in Leydig cells (15); however, the analysis used detected all isoforms of the leptin receptor and does not give information on its/their eventual functional properties. In this study, we were able to demonstrate that Leydig cells isolated from adult rat testis or derived from a mouse tumoral cell line express the long isoform of leptin receptor; rat Leydig cells, but not MLTC cells, coexpressed also the short Ob-Ra isoform. Because the leptin receptor was only detected by RT-PCR, which is a very sensitive method for mRNA detection, it is conceivable that the level of expression of leptin receptor in Leydig cells was low; however, the receptor was highly efficient and functional because, upon activation, it led to a rapid and dose-dependent inhibition of LH/hCG-stimulated T production. Interestingly, also in human ovarian cells, the signaling isoform of leptin receptor is expressed at levels detectable only by RT-PCR (24); in these cells, in analogy to results in rat Leydig cells, leptin has been found to inhibit LH-stimulated estradiol production, with no effects on basal steroid release. These results, together with other reports that showed that leptin has inhibitory actions on hormonally-stimulated ovarian steroidogenesis in vitro in different animal models (21, 22, 23), indicate that leptin, at proper levels, may signal metabolic information directly to the gonads via functional peripheral receptors besides the hypothalamic-pituitary (GnRH-FSH/LH) unit. Furthermore, the availability of a Leydig tumor cell line, i.e. MLTC-1 cells, expressing the functional leptin receptor is of great importance for future studies on leptin signal transduction pathway/s.
Several reports have addressed the association between leptin and circulating steroids in human males. It has been shown that leptin levels are lower in males, compared with females (44, 45, 46, 47); that hypogonadal men have higher circulating leptin, compared with hypogonadal patients under effective androgen substitution therapy (48); and that long-term exposure of human fat cells to T inhibits leptin expression in vitro (49). These results lead to the accepted knowledge that T is an important contributor to the gender difference in serum leptin levels. In our study, the inhibitory effect of leptin on hCG-stimulated T production was dose dependent and appeared at concentrations within the range of circulating levels in obese men. Obese subjects, as a group, have elevated levels of leptin in the blood and reduced androgen concentrations (50). It has been known for years that the entity of androgen reduction is related to the amount of fat mass (51) and, recently, that it is also related to leptin levels (52); in addition, we found that the androgen response to hCG stimulation is impaired in obese man, and multivariate analysis showed that leptin was the best hormonal predictor of the obesity-related reduction in androgen response (53). These observations, together with the present results, indicate that leptin excess may have an important role in the development of reduced androgens in male obesity. It is possible that leptin has a dualistic effect on male reproduction and that the major site of action may differ, depending on the concentration of leptin in the blood. The direct inhibitory action of leptin on the testis may be of importance under certain conditions, such as obesity (54, 55), with elevated plasma leptin/cerebrospinal fluid ratio and exposure of the peripheral tissues to very high leptin concentrations. Conversely, the positive leptin action at the hypothalamic level may be of relatively greater importance during conditions with low concentrations of leptin in the blood, as in subjects with low body mass index (6) and in ob/ob mice (5, 56, 57). It is also likely that leptin’s in vivo actions on Leydig cell steroidogenesis are more complex than those observed in vitro and could be influenced by various factors, including events that modify leptin concentrations (i.e. food restriction) and/or changes in hypotalamic peptides known to be modified by leptin and to regulate the HPG axis (i.e. POMC products, neuropeptide Y, galanin, and CRH) (13, 58).
The mechanism of leptin-induced inhibition of hCG-stimulated T production from rat Leydig cells was also investigated. The inhibitory actions of leptin were exerted after a short-time incubation (minutes) enough to reveal the action of other substances previously found to regulate Leydig cell function in a stimulatory (59) or inhibitory (60, 61) manner. Leptin markedly inhibited the androgen response, with no change in hCG sensitivity, suggesting a not-competitive mechanism of interaction between leptin and hCG; also, the observation that leptin inhibited 8-Br-cAMP-stimulated T production indicates that leptin action was exerted beyond cAMP. Interestingly, leptin significantly amplified hCG-induced cAMP formation both in MLTC-1 and rat Leydig cells, with no change in basal cAMP levels. These results were unexpected. The leptin receptor is known to stimulate JAK phosphorylation and in-sequence STAT protein phosphorylation, dimerization, translocation to the nucleus, and stimulation of transcription (38). It is believed that STAT3 and, maybe, STAT5 are activated by the triggering of Ob-R (39), and Stat5b protein has been recently found to be present and phosphorylated in rat Leydig cells and MA10 mouse Leydig tumor cell line after GH treatment (62). Thus, it is likely that, in Leydig cells, leptin stimulation of the Ob-R leads to the activation of the JAK/STAT pathway, which should be distinct from the adenylate cyclase-cAMP-PKA pathway. Explanations of leptin amplification of hCG stimulation of cAMP should exclude leptin-induced changes in phosphodiesterase activity, which has been shown to occur in other cells responsive to leptin (63), because all experiments were performed in the presence of adequate concentrations of MIX, a universal inhibitor of phosphodiesterases. Interestingly, we previously demonstrated that, in rat Leydig cells, CRF acutely inhibits hCG-stimulated T production (60), and that this is attributable to a rapid cross-talk between two distinct transducing signal pathways, i.e. the CRF-PKC and the hCG-adenylate cyclase pathways (61). It is possible that, in rodent cells, a positive interaction occurred between the JAK-STAT pathway and adenylate cyclase activity stimulated concomitantly by leptin and hCG. This possibility will be investigated in future studies.
The leptin-induced inhibition of hCG-stimulated androstenedione and T
production was explained by a lesion of 17,20-lyase activity, the
enzyme that converts 17-hydroxylated intermediates to
androstenedione (64), as shown by the increase in precursor metabolites
(pregnenolone, progesterone, and 17-OH-progesterone) and the augmented
percentage changes in precursor-to-product molar ratios
(17-OH-progesterone/androstenedione). Data obtained from MLTC-1 cells,
a tumor cell line not expressing cytochrome P450–17 (65), showed
that leptin, though causing a significant increase in hCG-stimulated
cAMP production, did not modify steroid release, further indicating
that leptin action occurs downstream of progesterone synthesis. In
other studies, long-term leptin incubation (1 day) has been found to
inhibit glucocorticoid release from bovine adrenocortical cells and to
significantly reduce ACTH-stimulated human and bovine adrenal
steroidogenesis through a transcriptional inhibition of cytochrome
P450–17 (25, 27). In our experiments, acute leptin treatment did
not cause any reduction in P450–17 mRNA accumulation, by Northern
blot analysis, indicating that its rapid action was not caused by mRNA
degradation but possibly by an inhibitory modulation of 17,20-lyase
activity. Along this possibility, we have previously shown that rapid
modifiers of hCG-stimulated Leydig cell function act via specific
alterations in the activity of cell enzymatic machinery (60, 61).
Further studies performed with prolonged treatment periods (24 h and
longer) to account for the half-life of the P450–17 message will be
needed to assess the effect of leptin on new P450–17 mRNA
synthesis. However, adult primary rat Leydig cells seem unsuitable for
this purpose, because these cells usually lose their LH/hCG receptor
after 1–3 days in culture along with a parallel decline of
17--hydroxylase/17,20-lyase activities (66).
The mechanism/s of action of leptin on 17,20-lyase activity is
presently unknown. In Leydig cells, the production of steroids in
response to hormone stimulation requires de novo protein
synthesis (67), but changes in phosphorylation of steroidogenic enzymes
also play an important role (68). In particular, serine phosphorylation
seems to be required exclusively for 17,20-lyase activity, as in human
adrenocortical cells, dephosphorylation of microsomal proteins with
alkaline phosphatase causes a complete loss of 17,20-lyase but no
change in 17-hydroxylase activity (69). Because a rapid increase in
phosphatase activity occurs after Ob-R activation (70), a
leptin-induced change in hormonally-stimulated phosphorylation of
specific P450–17 amino acids cannot be excluded. Finally, recent
reports showed that in vivo and in vitro
treatment with leptin activates c-fos gene expression in
specific hypothalamic nuclei (71, 72) and in stable transfected cells
(73), respectively. In Leydig cells, in contrast to hCG, which induced
a clear stimulation of c-fos expression, we were unable to
show any increase in c-fos mRNA levels by leptin, indicating
the lack of involvement of this early gene in leptin response.
In conclusion, the results of our study demonstrate that functional leptin receptors are expressed in rodent Leydig cells and that leptin has a novel direct negative action on LH/hCG-stimulated androgen production from Leydig cells in culture. These results are consistent with an endocrine action of leptin on the rat testis and have possible implications in some clinical aspects of male reproduction, such as the reduced testicular function in obese males.
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Acknowledgments |
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Footnotes |
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Received May 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA: the deduced amino-acid sequence, expression
and secondary structural configuration. Biochem Biophys Res Commun 157:705–712[CrossRef][Medline]
. Mol Endocrinol 5:1373–1380This article has been cited by other articles:
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L. R. Franca, M. O. Suescun, J. R. Miranda, A. Giovambattista, M. Perello, E. Spinedi, and R. S. Calandra Testis Structure and Function in a Nongenetic Hyperadipose Rat Model at Prepubertal and Adult Ages Endocrinology, March 1, 2006; 147(3): 1556 - 1563. [Abstract] [Full Text] [PDF]
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 S. Aquila, M. Gentile, E. Middea, S. Catalano, C. Morelli, V. Pezzi, and S. Ando Leptin Secretion by Human Ejaculated Spermatozoa J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4753 - 4761. [Abstract] [Full Text] [PDF]
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N. Pitteloud, M. Hardin, A. A. Dwyer, E. Valassi, M. Yialamas, D. Elahi, and F. J. Hayes Increasing Insulin Resistance Is Associated with a Decrease in Leydig Cell Testosterone Secretion in Men J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2636 - 2641. [Abstract] [Full Text] [PDF]
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 M. Zerani, C. Boiti, D. Zampini, G. Brecchia, C. Dall'Aglio, P. Ceccarelli, and A. Gobbetti Ob receptor in rabbit ovary and leptin in vitro regulation of corpora lutea J. Endocrinol., November 1, 2004; 183(2): 279 - 288. [Abstract] [Full Text] [PDF]
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 S. Bluher and C. S. Mantzoros The Role of Leptin in Regulating Neuroendocrine Function in Humans J. Nutr., September 1, 2004; 134(9): 2469S - 2474S. [Abstract] [Full Text] [PDF]
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 M. Caprio, E. Fabbrini, G. Ricci, S. Basciani, L. Gnessi, M. Arizzi, A. R. Carta, M. U. De Martino, A. M. Isidori, G. V. Frajese, et al. Ontogenesis of Leptin Receptor in Rat Leydig Cells Biol Reprod, April 1, 2003; 68(4): 1199 - 1207. [Abstract] [Full Text] [PDF]
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M. L. Barker-Gibb, A. Sahu, C. R. Pohl, and T. M. Plant Elevating Circulating Leptin in Prepubertal Male Rhesus Monkeys (Macaca mulatta) Does Not Elicit Precocious Gonadotropin-Releasing Hormone Release, Assessed Indirectly J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 4976 - 4983. [Abstract] [Full Text] [PDF]
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 F. Machinal-Quelin, M. N. Dieudonne, M. C. Leneveu, R. Pecquery, and Y. Giudicelli Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways Am J Physiol Cell Physiol, April 1, 2002; 282(4): C853 - C863. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere, M. L. Barreiro, L. C. Gonzalez, F. Gaytan, F.-P. Zhang, J. E. Caminos, L. Pinilla, F. F. Casanueva, C. Dieguez, and E. Aguilar Novel Expression and Functional Role of Ghrelin in Rat Testis Endocrinology, February 1, 2002; 143(2): 717 - 725. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere, L. Pinilla, F.-P. Zhang, L. C. González, I. Huhtaniemi, F. F. Casanueva, C. Dieguez, and E. Aguilar Developmental and Hormonal Regulation of Leptin Receptor (Ob-R) Messenger Ribonucleic Acid Expression in Rat Testis Biol Reprod, February 1, 2001; 64(2): 634 - 643. [Abstract] [Full Text]
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 G. Fantuzzi and R. Faggioni Leptin in the regulation of immunity, inflammation, and hematopoiesis J. Leukoc. Biol., October 1, 2000; 68(4): 437 - 446. [Abstract] [Full Text]
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T. El-Hefnawy, S. Ioffe, and M. Dym Expression of the Leptin Receptor during Germ Cell Development in the Mouse Testis Endocrinology, July 1, 2000; 141(7): 2624 - 2630. [Abstract] [Full Text] [PDF]
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A. M. Isidori, F. Strollo, M. Morè, M. Caprio, A. Aversa, C. Moretti, G. Frajese, G. Riondino, and A. Fabbri Leptin and Aging: Correlation with Endocrine Changes in Male and Female Healthy Adult Populations of Different Body Weights J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1954 - 1962. [Abstract] [Full Text]
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S. M. Hileman, D. D. Pierroz, and J. S. Flier Leptin, Nutrition, and Reproduction: Timing Is Everything J. Clin. Endocrinol. Metab., February 1, 2000; 85(2): 804 - 807. [Full Text]
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,
-hCG -leptin; 
, -hCG +leptin; 
, +hCG -leptin; 
, +hCG
+leptin; *, P < 0.05 level of significance; **,
P < 0.01 level of significance.
) or
presence (•) of leptin (150 ng/ml).
