0013-7227/99/$03.00/0
Endocrinology
Copyright © 1999 by The Endocrine Society
Vol. 140, No. 4
Printed in U.S.A.
Expression of a Leptin Receptor in Immortalized
Gonadotropin-Releasing Hormone-Secreting Neurons*
PAOLO MAGNI, ROBERTO VETTOR, CLAUDIO PAGANO,
ALESSANDRA CALCAGNO, ELENA BERETTA, ELIO MESSI, MARIAROSA ZANISI,
LUCIANO MARTINI, AND MARCELLA MOTTA
Institute of Endocrinology (P.M., E.B., E.M., M.Z., L.M., M.M.), University of Milan, Milan, Italy,
Institute of Semeiotica Medica (R.V., C.P., A.C.), University of Padua, Padua, Italy
ABSTRACT
Leptin is secreted by adipocytes and regulates food intake and
energy balance through the activation of specific receptors (OB-R).
Recent evidence suggests that it is also involved in the control of
reproductive processes, by possibly acting on central and peripheral
targets. In particular, it has been shown that leptin may indirectly
stimulate GnRH release from hypothalamic fragments by acting on
interneurons impinging on GnRH-secreting neurons. The possibility
that leptin might additionally modulate the activity of GnRH-secreting neurons in a direct way has been addressed in the present study,
by using the immortalized GnRH-secreting cell line GT1–7. The presence of OB-R messenger RNA (mRNA) (long form) was detected by
RT-PCR analysis of total RNA from GT1–7 cells. An OB-R protein is
also expressed in these cells, as shown by immunocytochemistry and
by Western blot analysis. The latter has revealed the presence of a
single immunoreactive OB-R with an approximate size of 130 kDa. To
L
EPTIN, the product of the ob gene, is a 16-kDa protein
produced and secreted by adipocytes (1) and is involved in the regulation of food intake and energy balance
(2). Leptin exerts its effects by interacting with specific membrane receptors (OB-R) (3). The multiple forms of OB-R,
which have been identified in different murine and human
tissues, include a long intracellular domain form (OB-Rb), a
group of short intracellular domain forms (OB-Ra, OB-Rc),
and a soluble form (OB-Re) lacking the transmembrane domain and probably representing a circulating binding protein for leptin (4 – 6). Recent studies have suggested that
leptin is also involved in the regulation of reproductive processes (7). Leptin treatment has been shown to restore fertility in mice with a genetic deficit of leptin (8, 9) and to
accelerate the onset of puberty in female rodents (10). However, the precise site(s) where leptin might exert its actions
on the reproductive system still remain(s) not completely
elucidated. The presence of OB-R mRNA and/or of leptin
binding sites in the rat ovary, testis, uterus, and placenta
suggests the possibility of peripheral actions of this hormone
Received July 28, 1998.
Address all correspondence and requests for reprints to: Dr. Paolo
Magni, Institute of Endocrinology, University of Milan, via Balzaretti 9,
20133 Milan, Italy. E-mail: [email protected].
* This work was supported by Grants ACRO 96.00594.PF39 from
Consiglio Nacionale delle Ricerche, by Associatione Italiana per la
Ricerca Sul Cancro, and by Ministero dell Università e della Ricerca
Scientifique. This work was presented in part at The Endocrine Society
Annual Meeting, 1998, New Orleans, Louisiana.
study the functionality of these receptors, the effect of leptin treatment on GnRH secretion and gene expression in GT1–7 cells were
evaluated. Under static conditions, GnRH release was stimulated by
exposure to low concentrations of leptin (10212 M after 30 min; 10210
212
M after 60 min). The 10
M dose was selected for studying the effect
of leptin on GnRH secretion under dynamic conditions. To this purpose, GT1–7 cells were placed in a perifusion system; treatment with
leptin (10212 M) for 60 min stimulated GnRH release with no changes
of pulse frequency. On the contrary, exposure to leptin (10212–10210
M) for 1, 3, 6, and 24 h did not affect GnRH gene expression in GT1–7
cells. The present results indicate that GT1–7 cells possess OB-Rs and
that leptin may directly affect their function. Taken together with the
available reports, these findings suggest that leptin might participate
in the regulation of reproductive processes by acting at multiple levels, both centrally and peripherally. (Endocrinology 140: 1581–1585,
1999)
(11, 12); however, evidence is available that indicates that
some actions of leptin on reproduction might derive from
influences on specific brain areas (9). High concentrations of
OB-R have been found in some hypothalamic nuclei implicated in the regulation of reproductive phenomena (4, 5, 13),
suggesting that these structures might be one target of the
reproductive actions of leptin. Recent studies have postulated that leptin may facilitate the secretion of GnRH from
hypothalamic explants via indirect mechanisms, i.e. by acting
on interneurons impinging on GnRH secreting cells (14, 15).
In the present study, we have evaluated whether, in addition
to the indirect effects mentioned above, leptin might also
directly affect GnRH synthesis and/or secretion, acting via
its own receptors. Since the presence of OB-R mRNA has
been reported in GT1–7 cells (11), a mouse-derived clone of
immortalized GnRH-secreting neurons, we have taken advantage of this in vitro model to assess the presence of an
OB-R protein in these cells and its possible functionality in
terms of regulation of GnRH release and gene expression.
Materials and Methods
Culture of GT1 cells
GT1–7 cells (a kind gift of Dr. R. I. Weiner, San Francisco, CA) were
maintained in DMEM-4.5 mg/liter glucose (Biochrom, Berlin, Germany)
supplemented with 10% FCS (Life Technologies, Grand Island, NY), 100
U/ml penicillin, and 100 mg/ml streptomycin. For the study of GnRH
release under static conditions, GT1–7 cells were plated in 12-well plates;
the experimental conditions used for the study of GnRH release under
dynamic conditions are reported below. For the study of GnRH gene
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1582
LEPTIN RECEPTOR AND GnRH-SECRETING NEURONS
expression, GT1–7 cells were plated in 100 mm Petri dishes. Mouse leptin
was obtained from Linco Research, Inc. (St. Charles, MO).
Immunocytochemistry
The presence of OB-R immunoreactivity on GT1–7 cells was studied
by immunocytochemistry (ICC). Cells were grown on glass slides, fixed
in acetone-chloroform solution. Fixed GT1–7 cells were preincubated for
15 min in normal rabbit serum, and then incubated for 30 min in a leptin
receptor antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
diluted 1:100. Cells were washed three times in PBS for 5 min, incubated
for 30 min with the secondary antibody and washed in PBS. The immunocytochemical visualization was performed by peroxidase antiperoxidase complex. The chromogen diaminobenzidine was added for 5
min. All passages were at RT. In our experiments, control studies were
performed using the above methods, with deletion of individual primary or secondary antisera. At the end, GT1–7 cells were stained with
hematoxylin and eosin and mounted in synthetic resin.
Western blotting
GT1–7 cells were washed in PBS and resuspended in Laemmli sample
buffer. Cells were dissolved by vigorous shaking and stored at 280 C
for subsequent immunoblot analysis of OB-R, using the same antiserum
used for ICC. Protein samples and prestained molecular mass markers
(Amersham Italia, Milan, Italy) were separated on a 8% SDS-polyacrylamide gel. Proteins were transferred from the gel to a nitrocellulose filter
paper. Membrane was incubated with a diluted solution of the primary
antibody (1:3,000) and with a second antibody conjugated with peroxidase (1:80,000; Sigma-Aldrich Co., Milan, Italy). OB-R was detected by
the ECL method and exposure of the membrane to autoradiographic
film (Hyperfilm; Amersham Italia) at RT. Band intensity appeared to be
linear between 5 and 60 mg of protein and the blot obtained with 20 mg
is shown. A negative control carried out by incubating the membrane
with either a preimmune serum or the second antibody alone did not
result in any band on the blot.
Perifusion of immortalized GnRH neurons
GT1–7 cells were cultured as indicated above and then grown on
Cytodex-3 beads (Pharmacia Biotech, Uppsala, Sweden) (16). After 3– 4
days, cells were loaded into temperature-controlled glass syringes, the
final cell-matrix volume was adjusted to 0.15 ml. Chambers were perifused at a flow rate of 10 ml/h with Locke’s medium (mm): NaCl 154;
KCl 5.6; CaCl2 2.2; NaHCO3 6; glucose 10; HEPES 2; pH 7.4; gassed with
95% O2–5% CO2 at 37 C (17). After a 2-h equilibration period, samples
were collected every 90 sec and stored at 220 C until radioimmunoassayed for GnRH. Cells were perifused for the first hour with Locke’s
medium, and then with medium containing leptin (10212 m) for another
hour. GnRH pulses were identified and their parameters were determined by a computer algorithm cluster analysis (18). The occurrence of
pulses and the duration of pulses are shown diagrammatically above
each plot.
Endo • 1999
Vol 140 • No 4
manufacturer’s recommendations. Primers designed for a high-homology region of the mouse and human leptin receptor complementary
DNA (cDNA) were used (forward primer: 59-ATg ACg CAg TgT ACT
gCT g-39; reverse primer: 59-gTg gCg AgT CAA gTg AAC CT-39). The
PCR reaction, carried out in a DNA thermal cycler (Perkin Elmer Corp.),
included an initial denaturing step at 94 C for 3 min, 35 cycles (94 C/1
min 1 62 C/1 min 1 72 C/1 min), and a final step at 72 C for 10 min.
Control RNA (pAW109RNA) included in the kit was subjected to the
same RT-PCR reaction, according to the manufacturer. The amplified
products were resolved in a 2% agarose/1X TBE gel, and the DNA was
visualized by ethidium bromide fluorescence on a UV transilluminator.
GnRH gene expression was evaluated by Northern blot analysis. Twenty
micrograms of each total RNA sample were electrophoresed on denaturating 1.2% agarose gel. The fractionated RNA was blotted onto a
Nytran membrane (Schleicher & Schuell, Düren, Germany) and hybridized for GnRH mRNA and 28sRNA (control) with 32P RNA probes. The
plasmid SP65, containing DNA fragments representing rat GnRHcDNA (generously provided by Prof. P. H. Seeburg), and the plasmid
pGEM-3Z, containing the cDNA for the 28s, were linearized and transcribed using SP6 RNA polymerase (Boehringer Mannheim Italia, Milan,
Italy) and 32P-UTP (Amersham Italia) to obtain respectively the GnRH
and the 28s-probe. Hybridizations were carried out for 18 h at 60 C in
standard hybridization solution in the presence of 50% formamide.
Filters were washed with 1X standard saline citrate (SSC)/0.1% SDS for
15 min at RT and with 0.1X SSC/0.1% SDS for 1h at 67 C. Hybridization
signals were quantitated by scanning the autoradiographic films with an
Apple OneScanner densitometer.
Statistics
Data relative to GnRH gene expression and to GnRH release (static
incubation) were analyzed by ANOVA, using Tukey’s test for multiple
comparisons, by using the Systat package (Systat, Evanston, IL) on an
Apple Macintosh computer.
Results
Expression of OB-R in GT1–7 cells
Figure 1 shows the results of RT-PCR analysis of the gene
expression of OB-R in GT1–7 cells. In total RNA extracts from
these cells, as well as in mouse hypothalamic extracts (positive control), a PCR product of 356 bp was detected, indicating the presence of OB-R mRNA. This experiment is in
RIA
The concentration of GnRH in the media and in the fractions collected
during the perifusion experiments was determined by RIA using a rabbit
antibody (Sigma-Aldrich Co.) and iodinated GnRH (Amersham Italia).
The GnRH standard was from NovaBiochem (Laufelfingen, Switzerland). All samples were run in duplicate; the detection limit was 3.9
pg/ml. The inter and intraassay coefficients of variation were 9.4% and
6.6%, respectively. Each experiment was repeated at least three times.
Total RNA extraction, RT-PCR, and Northern blot analysis
Total cellular RNA was extracted with the guanidine/chloroform
method (19). RT-PCR for the detection of OB-R mRNA was performed
according to Zamorano et al. (11). One microgram of total RNA from
GT1–7 cells or from mouse hypothalamus was incubated with 50 ng of
random hexamers at 70 C for 10 min. The RT reaction using MuLV-RT
was carried out in a 20 ml volume, using a commercially available
RT-PCR kit (Perkin Elmer Corp., Foster City, CA) and following the
FIG. 1. RT-PCR analysis for the expression of the leptin receptor
(OB-R) gene in GT1–7 cells and in mouse hypothalamus (positive
control). Note the presence of a PCR product of the appropriate size
in GT1–7 and hypothalamic samples in the presence (1 MuLV-RT) of
MuLV-RT and its absence, when this enzyme was not added (no
MuLV-RT), indicating that there was no contamination by genomic
DNA. pAW109RNA from the RT-PCR kit was used as internal control
of the RT-PCR reaction.
LEPTIN RECEPTOR AND GnRH-SECRETING NEURONS
agreement with previously reported findings in these cells
(11). Note that, in the absence of RT, no expression of OB-R
was observed, indicating that the samples were not contaminated by genomic DNA. In addition, after a GenBank search,
it was found that the oligoprimers used in this RT-PCR
reaction can match only the sequence with accession n°
U49107/U46135, corresponding to OB-Rb (the long isoform
of the receptor), but not the sequences with accession number
AF039459, AF039460, AF039456, and AF039461 (corresponding to OB-Rc, OB-Ra, OB-Re and OB-Rb-short, respectively).
These observations suggest that the OB-R mRNA detected in
GT1–7 cells by using these primers should correspond to the
long form of this receptor.
The presence of the OB-R protein in GT1–7 cells was evaluated by immunocytochemistry and Western blot analysis.
The immunocytochemical analysis of the cellular distribution of OB-R indicated that a high percentage (about 70 – 80%)
of GT1–7 cells were immunopositive, with a whole-cell staining (membrane and cytoplasm) (Fig. 2). GT1–7 cell extracts
were subjected to immunoblot analysis with an anti-OB-R
antibody. As shown in Fig. 3 (panel A), the major form of a
OB-R protein that is expressed in GT1–7 cells and in mouse
hypothalamus (positive control) possesses a molecular mass
of approximately 130 kDa, as suggested by the detection of
a single immunoreactive band.
1583
FIG. 3. Western blot analysis of OB-R in GT1–7 cells. Membranes
were incubated with an OB-R antibody (A) or a nonimmune goat
serum (B). A major band of the size of about 130 kDa was detected both
in the hypothalamus and in the GT1–7 cell line.
Leptin regulation of GnRH secretion and gene expression in
GT1–7 cells
The possible functionality of the OB-Rs present in GT1–7
cells was first assessed by studying the effect of leptin treatment on GnRH release. In experiments conducted under
static conditions, GT1–7 cells were treated with leptin (10214–
1028 m) for 30 and 60 min (Fig. 4). This treatment induced a
significant stimulation of GnRH release at the dose of 10212
m after 30 min. After a 60-min exposure to leptin, GnRH
secretion was stimulated in a dose-dependent manner,
reaching a plateau at the concentration of 10210 m (Fig. 4).
An additional characterization of the dynamics of the
modulatory action of leptin on GnRH secretion was then
performed using a perifusion system. Basal release of GnRH
by GT1–7 cells was intrinsically pulsatile (Fig. 5), as detected
FIG. 2. Immunocytochemical identification of OB-R in GT1–7 cells.
Most cells are immunopositive, with a whole-cell pattern of staining.
FIG. 4. Effect of leptin on the release of GnRH by GT1–7 cells (static
incubation). GT1–7 cells were treated with leptin (10214–1028 M) for
30 or 60 min. GnRH released into the medium was assayed by RIA.
Data are expressed as mean 6 SEM (n 5 5– 6). *, P , 0.05 vs. control
(Tukey test).
by the cluster analysis algorithm, and according to previous
reports (16). After 1 h of perifusion under basal conditions,
leptin (the dose of 10212 m was selected from the experiments
performed under static conditions) was added to the perifusion medium. This treatment was carried on for 60 min and
resulted in a stimulation of GnRH release, without changes
of the frequency of the secretory peaks (Fig. 5). The baseline
secretory activity of GT1–7 cells, as well as their response to
leptin were quantitatively different from one perifusion experiment to another (due to intrinsic features of this system);
however, the effect of leptin was qualitatively similar.
Because OB-R activation leads to the stimulation of intracellular signals, like the JAK-STAT and the mitogen-activated protein kinase (MAPK) pathways, able to influence
gene expression, the effect of leptin treatment on GnRH gene
expression in GT1–7 cells was evaluated. Cells were treated
for 1, 3, 6, or 24 h with leptin (10212–10210 m), total RNA was
extracted, and GnRH gene expression was evaluated by
Northern blot analysis. As shown in Fig. 6, leptin treatment
did not affect GnRH mRNA levels at all times considered;
only a slight, but not significant increase was detectable at 1 h
at the dose of 10212 m.
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LEPTIN RECEPTOR AND GnRH-SECRETING NEURONS
Endo • 1999
Vol 140 • No 4
FIG. 6. Effect of leptin on GnRH gene expression in GT1–7 cells. Cells
were treated with leptin (10212 or 10210 M) for 1, 3, 6, and 24 h. The
data shown are referred to densitometric analysis of a representative
Northern blot analysis and are expressed as mean 6 SEM of the
GnRH-mRNA/28sRNA ratio; n 5 3– 4. Differences were statistically
not significant (ANOVA test).
FIG. 5. Effect of leptin on the release of GnRH by GT1–7 cells (perifusion). GT1–7 cells, grown on Cytodex-3 beads, were placed in a
perifusion apparatus and exposed (horizontal bar) to 10212 M leptin
for 60 min. The GnRH content in each fraction of the perifusate was
quantified by RIA. The results of two not simultaneous experiments,
representative of four separate experiments, are shown. The segmented bar on top of the profile of GnRH indicates the position and
duration of secretory pulses. Above each graphic representation of
pulses the area under each secretory peak, as calculated by the Cluster algorithm is indicated. Note the scale difference between profiles.
Discussion
The present results show that GT1–7 cells, an immortalized cell line of mouse GnRH-secreting neurons, express an
OB-R protein, which is localized on both the plasma membrane and cytoplasm and possesses a molecular mass of
about 130 kDa. These results seem to disagree with a recent
immunocytochemical study, reporting that the presence of
OB-R was not found in GnRH-expressing neurons in the
hypothalamus of male rat (20). However, as these authors
comment, one cannot exclude low levels of OB-R in these
cells, not detectable by immunocytochemistry. The present
data, obtained at the mRNA (RT-PCR) and protein (Western
analysis) level, suggest that the OB-R isoform expressed in
GT1–7 cells is the long form (OB-Rb), which is the one predominantly expressed in the hypothalamus (21). The OB-Rb
is considered the receptor form able to activate intracellular
signals upon binding with its ligand (22). However, the second messenger system linked to the OB-R present in GT1–7
cells has not been clarified so far. On the basis of the data
available in other systems, candidate signals through which
leptin might stimulate GnRH release in GT1–7 cells are the
JAK-STAT pathway, which is linked to OB-R in some cells,
as well as other mechanisms, like the MAPK and the phosphatidylinositol 3-kinase pathways, shown to be triggered by
some class I cytokine receptors (23, 24). In addition, recent
data indicate that also the short form OB-Ra might possess
some signalling capabilities (25). The OB-R present in GT1–7
cells appears to be functional, since treatment of these cells
with leptin stimulated the release of GnRH, but did not affect
the expression of the GnRH gene. Leptin was active acutely,
after 30 – 60 min of incubation, at concentrations in the picomolar range and able to induce rather moderate, although
significant increments. Leptin increased the secretion of
GnRH with no changes of its pulsatility pattern. These data
are consistent with the timing, the effective doses, and the
extent of stimulation reported on leptin-induced GnRH release from hypothalamic fragments (14). It is possible that the
rather small effect of leptin or the lack of effect at higher doses
(in the present and in other studies) might also result from
down-regulation of the receptor and/or the signalling
pathway.
The present data on the direct effect of leptin on GnRH
secreting neurons complete and integrate those of other authors (14, 15), who suggest the possibility that leptin might
modulate the secretion of GnRH by indirect influence at the
hypothalamic levels. Thus, the net effect of leptin on this
process would derive from the modulation of GnRH-secreting nerve terminals present in the median eminence, as well
as of interneurons producing transmitters such as neuropeptide Y, POMC-derived peptides, nitric oxide and norepinephrine (13–15, 26). It is interesting to note that the median
eminence of the hypothalamus is a structure lacking the
blood-brain barrier: leptin might thus act on the GnRHsecreting nerve endings contained in it without needing a
transport mechanism, as, on the contrary, has been hypothesized for its actions on other hypothalamic areas (5, 27, 28).
It is well known that GnRH release is controlled by a
complex and redundant hormonal system. In this context,
the available literature and the present study suggest that
leptin, more than just one specific controller of GnRH release,
might be a modulator of reproductive function at the GnRH
levels as well as at other levels (pituitary, gonads) of this axis,
LEPTIN RECEPTOR AND GnRH-SECRETING NEURONS
probably interacting with other agents and facilitating their
action (13, 14, 26). Plasma levels of leptin seem rather constant; however, they show important variations in relationship to the the state of gonadal function and the consequent
levels of circulating sex steroids (29). It is possible that such
variations of plasma leptin might in turn influence the GnRH
secreting system (neurons and interneuron).
On the basis of the present data, showing the expression
of an OB-R protein in GT1–7 cells and its functionality in
directly modulating GnRH release, and together with the
large body of literature available, it appears that leptin might
be involved in the regulation of reproductive processes at
central (with direct and indirect effects on GnRH neurons)
and peripheral sites.
Acknowledgments
The authors thank Ms. Paola Assi, Ms. Giovanna Miccichè, and Ms.
Ornella Mornati for their skillful technical collaboration.
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