Endocrinology Vol. 140, No. 4 1581-1585
Copyright © 1999 by The Endocrine Society
Expression of a Leptin Receptor in Immortalized Gonadotropin-Releasing Hormone-Secreting Neurons1
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
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: magni{at}imiucca.csi.unimi.it
 |
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 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 (10-12 M after 30
min; 10-10 M after 60 min). The
10-12 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 (10-12 M) for 60 min stimulated
GnRH release with no changes of pulse frequency. On the contrary,
exposure to leptin (10-12–10-10
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.
|
Introduction
|
|---|
LEPTIN, 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, 5, 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 (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 µg/liter glucose
(Biochrom, Berlin, Germany) supplemented with 10% FCS (Life Technologies, Grand Island, NY), 100 U/ml penicillin, and 100
µg/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
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
-80 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 µg of protein and the blot obtained with 20 µg 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
-20 C until radioimmunoassayed for GnRH. Cells were perifused for the
first hour with Locke’s medium, and then with medium containing leptin
(10-12 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.
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 µl volume, using a commercially available
RT-PCR kit (Perkin Elmer Corp., Foster City, CA) and
following the manufacturer’s recommendations. Primers designed for a
high-homology region of the mouse and human leptin receptor
complementary DNA (cDNA) were used (forward primer: 5'-ATg ACg CAg TgT
ACT gCT g-3'; reverse primer: 5'-gTg gCg AgT CAA gTg AAC CT-3'). 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 + 62 C/1 min + 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 GnRH-cDNA (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 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.
View larger version (51K):
[in this window]
[in a new window]
|
Figure 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 (+
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.
|
|
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.
View larger version (84K):
[in this window]
[in a new window]
|
Figure 2. Immunocytochemical identification of OB-R in
GT1–7 cells. Most cells are immunopositive, with a whole-cell pattern
of staining.
|
|
View larger version (97K):
[in this window]
[in a new window]
|
Figure 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 (10-14–10-8
M) for 30 and 60 min (Fig. 4). This treatment induced a significant
stimulation of GnRH release at the dose of 10-12
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 10-10 M (Fig. 4).
View larger version (36K):
[in this window]
[in a new window]
|
Figure 4. Effect of leptin on the release of GnRH by GT1–7
cells (static incubation). GT1–7 cells were treated with leptin
(10-14–10-8 M) for 30 or 60 min.
GnRH released into the medium was assayed by RIA. Data are expressed as
mean ± SEM (n = 5–6). *, P
< 0.05 vs. control (Tukey test).
|
|
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 by the
cluster analysis algorithm, and according to previous reports (16).
After 1 h of perifusion under basal conditions, leptin (the dose
of 10-12 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.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 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 10-12 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.
|
|
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
(10-12–10-10 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 10-12 M.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 6. Effect of leptin on GnRH gene expression in GT1–7
cells. Cells were treated with leptin (10-12 or
10-10 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 ±
SEM of the GnRH-mRNA/28sRNA ratio; n = 3–4.
Differences were statistically not significant (ANOVA test).
|
|
|
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, 14, 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 GnRH-secreting 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, 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.
|
Footnotes
|
|---|
1 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. 
Received July 28, 1998.
|
References
|
|---|
-
Zhang Y, Proenca R, Maffei M, Baroni M, Leopold L,
Friedman JM 1994 Positional cloning of the mouse obese gene and
its human homologue. Nature 372:425–432[CrossRef][Medline]
-
Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters
D, Boone T, Collins F 1995 Effects of the obese gene product on
body weight regulation in ob/ob mice. Science 269:540–543[Abstract/Free Full Text]
-
Tartaglia LA, Demski M, Weng X, Deng N, Culpepper J,
Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker
S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA,
Tepper RI 1995 Identification and expression cloning of a leptin
receptor, OB-R. Cell 83:1263–1271[CrossRef][Medline]
-
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah
LT, Morgan PJ, Trayhurn P 1996 Coexpression of leptin receptor and
preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus.
J Neuroendocrinol 8:733–735[CrossRef][Medline]
-
Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin
DG 1996 Identification of targets of leptin action in rat
hypothalamus. J Clin Invest 98:1101–1106[Medline]
-
Tartaglia LA 1997 The leptin receptor. J Biol
Chem 272:6093–6096[Free Full Text]
-
Mounzih K, Lu R, Chehab FF 1997 Leptin treatment
rescues the sterility of genetically obese ob/ob males. Endocrinology 138:1190–1193[Abstract/Free Full Text]
-
Chehab F, Lim M, Ronghua L 1996 Correction of the
sterility defect in omozygous obese female mice by treatment with the
human recombinant leptin. Nat Genet 12:318–320[CrossRef][Medline]
-
Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB,
Kuijper JL, Clifton DK, Steiner RA 1996 Leptin is a metabolic
signal to the reproductive system. Endocrinology 137:3144–3147[Abstract]
-
Chehab FF, Mounzih K, Lu R, Lim ME 1997 Early onset
of reproductive function in normal female mice treated with leptin.
Science 275:88–90[Abstract/Free Full Text]
-
Zamorano PL, Mahesh VB, Sevilla LMD, Chorich LP, Bhat
GK, Brann DW 1997 Expression and localization of the leptin
receptor in endocrine and neuroendocrine tissues of the rat.
Neuroendocrinology 65:223–228[CrossRef][Medline]
-
Hoggard N, Hunter L, Duncan JS, Williams LM, Trayhurn P,
Mercer JG 1997 Leptin and leptin receptor mRNA and protein
expression in the murine fetus and placenta. Proc Natl Acad Sci USA 94:11073–11078[Abstract/Free Full Text]
-
Kalra SP, Kalra PS 1996 Nutritional infertility:
the role of the interconnected hypothalamic neuropeptide
Y-galanin-opioid network. Front Neuroendocrinol 17:371–401[CrossRef][Medline]
-
Yu WH, Kimura M, Walczewska A, Karanth S, McCann SM 1997 Role of leptin in hypothalamic-pituitary function. Proc Natl Acad
Sci USA 94:1023–1028[Abstract/Free Full Text]
-
Yu WH, Walczewska A, Karanth S, McCann SM 1997 Nitric oxide mediates leptin-induced luteinizing hormone-releasing
hormone (LHRH) and LHRH and leptin-induced LH release from the
pituitary gland. Endocrinology 138:5055–5058[Abstract/Free Full Text]
-
Wetsel WC, Valenca MM, Merckenthaler I, Liposits Z,
Lopez FJ, Weiner RI, Mellon PL, Negro-Vilar A 1992 Intrinsic
pulsatile secretory activity of immortalized luteinizing
hormone-releasing hormone-secreting neurons. Proc Natl Acad Sci USA 89:4149–4153[Abstract/Free Full Text]
-
Escalera GMdl, Choi ALH, Weiner RI 1994 Biphasic
GABAergic regulation of GnRH secretion in GT1 cell lines.
Neuroendocrinology 59:420–425[Medline]
-
Veldhuis JD, Johnson ML 1986 Cluster analysis: a
simple, versatile and robust algorythm for endocrine pulse detection.
Am J Physiol 250:E486–E493
-
Chomczinski P, Sacchi N 1987 Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal Biochem 162:156–159[Medline]
-
Hakansson M-L, Brown H, Ghilardi N, Skoda RC, Meister
B 1998 Leptin receptor immunoreactivity in chemically defined
target neurons of the hypothalamus. J Neurosci 18:559–572[Abstract/Free Full Text]
-
Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH,
Skoda RC 1996 Defective STAT signaling by the leptin receptor in
diabetic mice. Proc Natl Acad Sci USA 93:6231–6235[Abstract/Free Full Text]
-
Ghilardi N, Skoda RC 1997 The leptin receptor
activates Janus Kinase 2 and signals for proliferation in a
factor-dependent cell line. Mol Endocrinol 11:393–399[Abstract/Free Full Text]
-
Kishimoto T, Taga T, Akira S 1994 Cytokine signal
transduction. Cell 76:253–262[CrossRef][Medline]
-
Heldin HC 1995 Dimerization of cell surface
receptors in signal transduction. Cell 80:213–223[CrossRef][Medline]
-
Yamashita T, Murakami T, Otani S, Kuwajima M, Shima
K 1998 Leptin receptor signal transduction: OBRa and OBRb of fa
type. Biochem Biophys Res Commun 246:752–759[CrossRef][Medline]
-
Thornton JE, Cheung CC, Clifton DK, Steiner RA 1997 Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob
mice. Endocrinology 138:5063–5066[Abstract/Free Full Text]
-
Cioffi J, Shafer A, Zupancic T, Smith-Gbur J, Mikhail A,
Platika D, Snodgrass H 1996 Novel B219/OB receptor isoforms:
possible role of leptin in hematopoiesis and reproduction. Nature Med 2:585–588[CrossRef][Medline]
-
Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM 1996 Leptin enters the brain by a saturable system independent of
insulin. Peptides 17:305–311[CrossRef][Medline]
-
Rosenbaum M, Nicolson M, Hirsch J, Heymsfield SB,
Gallagher D, Chu F, Leibel RL 1996 Effects of gender, body
composition, and menopause on plasma concentrations of leptin. J
Clin Endocrinol Metab 81:3424–3427[Abstract]
Top
Abstract
Introduction
