Download effect of leptin on thyroid-stimulating hormone secretion and nitric

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2014, 65, 1, 145-151
www.jpp.krakow.pl
P. RADWANSKA, U. KOSIOR-KORZECKA
EFFECT OF LEPTIN ON THYROID-STIMULATING HORMONE SECRETION
AND NITRIC OXIDE RELEASE FROM PITUITARY CELLS OF EWE LAMBS IN VITRO
Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, Lublin, Poland
Taking into account that the relationship between leptin and thyroid-stimulating hormone (TSH) secretion in ewe lambs
is not clear, the objective of the present study was to estimate the effect of leptin on basal TSH secretion from ovine
pituitary cells in vitro. Moreover, the influence of leptin on nitric oxide (NO) release and its role in the leptin-modulated
secretion of TSH was studied. Pituitary cells were cultured in the McCoy 5A medium without hormones (the control),
with 10–10–10–5 mol/L of leptin or with 10–10–10–5 mol/L of leptin and L-NAME (Nw-nitro-L-arginine methyl ester,
3×10–4 mol/L, the inhibitor of NO synthesis). The secretion of thyroid-stimulating hormone as well as concentration of
nitrite (as an indicator of nitric oxide release) were analysed after 2–72 hours of experiment. The obtained results show
that TSH secretion from ovine pituitary cells in vitro is dependent on leptin concentration: 10–10–10–6 mol/L of leptin
causes an increase in TSH secretion, whereas the highest concentration of leptin (10–5 mol/L) suppresses thyroidstimulating hormone release compared to the control. TSH secretion reaches the highest values under the influence of
10–8 mol/L of leptin. Moreover, leptin, depending on its dose and time of cell exposition, stimulates NO release from
anterior pituitary cells of ewe lambs in vitro. The inhibition of NO synthesis with L-NAME annihilates the stimulating
effect of leptin on thyroid-stimulating hormone secretion.
K e y w o r d s : leptin, thyroid-stimulating hormone, nitric oxide, ewe lambs, pituitary cells, nitric oxide synthase, Nw-nitro-Larginine methyl ester
INTRODUCTION
Thyroid hormones as well as leptin are involved in regulation
of energy metabolism, thermogenesis, food intake, glucose and
lipid metabolism as well as oxidation of fatty acids (1-8).
Moreover, leptin plays the significant role in neuroendocrine
functions and initiates the activity of the hypothalamic-pituitaryovarian axis (9). By means of Ob-Rb (long form) and Ob-Ra
(short isoform) receptors, it affects the neurons in the arcuate
nucleus of the hypothalamus (inducing the GnRH secretion) (10)
and influences gonadotropic cells (increasing the FSH and LH
secretion) (11). A few literature reports indicate that also
hormones of thyrotropic axis participate in the regulation of
reproductive processes in ruminants. In cattle, it has been shown
that the ovarian follicular fluid contains thyroid hormones
involved in the maturation of ovarian follicles (12, 13). According
to Spicer et al. (14), thyroid hormones may exert a stimulatory
effect on ovarian functions by acting on ovarian granulose and
theca cells. There are some studies on the contribution of leptin to
the modulation of thyroid-stimulating hormone (TSH) secretion in
rodents. It has been demonstrated that leptin stimulates TSH
secretion in rats (15). However, according to Ortiga-Carvalho et
al. (16), in vitro leptin inhibits release of thyroid-stimulating
hormone from male rats pituitary cells, while it has a stimulatory
effect on the TSH secretion in vivo. Therefore, these reports are
ambiguous and do not relate to sheep. Taking into account the fact
that the relationship between leptin and thyroid-stimulating
hormone secretion in ewe lambs is not clear, the first aim of the
study was to analyse the influence of leptin on TSH secretion.
Nitric oxide (NO) is a free radical, which is synthesized from
L-arginine and molecular oxygen by nitric oxide synthases
(NOS). It has a wide spectrum of cell functions and plays a
significant role in many biological processes, e.g. as a mediator
of immunity, modulator of neuronal activity, vasodilator,
regulator of blood flow or blood pressure (17-22). Nitric oxide
participates also in the modulation of endocrine system. Taking
into account the localization of cNOS in the pituitary gland, the
secretion of tropic hormones from anterior pituitary cells can be
mediated by NO (23-25). It is known that influence of leptin on
gonadotropin secretion in ovine pituitary cells can be mediated
by nitric oxide (26, 27). There are also reports that secretion of
TSH can be associated with the NO system. According to Coiro
et al. (28), TRH stimulates TSH secretion via nitric oxide in
human. However, there are also some studies showing that NO
exerts the inhibitory effect on TSH secretion in female rats (29).
These reports are contradictory and relate to human and rodents,
whereas, there is no similar data with regard to regulation of
thyroid-stimulating hormone secretion in ewes. Therefore, in the
second part of this study we examined the effect of leptin on NO
release and its role in the leptin-modulated secretion of TSH
under the condition of normal and inhibited nitric oxide
synthesis in ovine pituitary cells in vitro.
146
MATERIAL AND METHODS
The protocol of the study concept and all procedures were
approved by the Second Lublin Local Ethics Committee for
Animal Experimentation.
Pituitary glands were obtained immediately after slaughter
from the ewe lambs of the SCP line (50% Suffolk + 25%
Romanov + 25% Polish Lowland Sheep), aged 10 months, which
were selected on the basis of the ovarian activity (follicular phase,
verified by laparoscopic method). The presented study was
carried out on three independent cell cultures. Each cell culture
was prepared using pituitaries isolated from 7 ewe lambs.
Isolation of cells was carried out through the digestion of the
pituitaries with 0.25% trypsin solution. Suspension of cells in
trypsin combined with the preparatory medium (DMEM
supplemented with 0.1% BSA, 0.08% glucose, 0.59 HEPES, and
gentamycin with the final concentration of 20 µg/mL) was
centrifuged (1200 rpm for 10 min). The sedimented cells were
washed twice and suspended in the McCoy 5A medium
containing 2.5% fetal calf serum, 10% horse serum, mixture of
amino acids and vitamins, 0.59% HEPES, gentamycin (20
µg/mL), and adjusted to pH 7.4 (30-32). One millilitre of
suspension containing 250,000/mL was transferred to each well
of 24-well culture plates and incubated for 72 hours at 37°C
under the atmosphere of 5% CO2. After attachment to the dishes,
the cells were washed and finally cultured in the McCoy 5A
medium without hormones (the control), with 10–10–10–5 mol/L of
leptin or with 10–10–10–5 mol/L of leptin and L-NAME ( N wnitro-L-arginine methyl ester, 3×10–4 mol/L, the inhibitor of NO
synthesis), respectively. Each sample was prepared in duplicate.
After 2, 24, 48 and 72 hours of incubation the media for
thyroid-stimulating hormone and nitric oxide analysis were
collected and the proliferation index (PI) of the control cells and
those treated with leptin was determined. The results were used
for calculation of TSH secretion. Assessment of cell
proliferation was based on the reduction of tetrazolium salt
(MTT) into a blue formazan. The control cultures and those
incubated with leptin were pulsed with 15 µl of MTT (for 3
hours at 37°C) and then solubilized with 10% solution of sodium
dodecyl sulphate (SDS) overnight. The optical density (OD) of
the formed blue formazan was measured by ELISA microplate
reader at the wavelength of 600 nm. The results were expressed
as PI values. TSH concentration in the culture medium was
determined using Sheep Thyroid Stimulating Hormone ELISA
KIT (Blue Gene, Shanghai, China). TSH secretion was
expressed as a concentration (µIU/mL) of hormone which was
released into the culture medium by 250,000 cells for 2, 24, 48
or 72 hours, respectively. Concurrently, concentration of nitrite
(NO2–) as an indicator of nitric oxide (NO) production was
measured. Equal amounts of the sample and the Griess reagent
(sulfanilamide 2% (w/v), N-(1-naphtyl)ethylenediamine 0.2%
(w/v), phosphoric acid 4% (v/v)) were mixed. After 10 min of
incubation at room temperature the absorbance at 545 nm was
measured. As the standard NaNO2 was used (25, 26, 30).
Statistical analysis
The obtained results were calculated using Statistica 5.0 PL
and expressed as mean and standard deviation, x ± S.D.
Comparisons between the control and the experimental cultures
were performed using the analysis of variance and the paired
t-tests. Differences were considered as significant at P£0.05.
RESULTS
Influence of leptin on thyroid-stimulating hormone secretion
from ovine pituitary cells in vitro
The basal secretion of TSH amounted from 1.27 ± 0.02 to
1.31 ± 0.06 µIU/mL/250,000 cells during the whole time of the
experiment (0–72 hours). The effect of leptin on TSH secretion
Fig. 1. TSH secretion
from ovine pituitary
cells in vitro under the
influence of leptin in
the concentration of
10–10–10–5 mol/L.
*- significant difference
compared to the control
(P£0.05)
147
by ovine pituitary cells was dependent on time and the dose of
leptin used. The introduction of leptin in the concentration 10–10
to 10–5 mol/L affected TSH secretion compared to the control
after 24, 48, 72 hours. After 2 hours, thyroid-stimulating hormone
secretion did not change significantly. The addition of 10–10
mol/L of leptin to the culture medium resulted in slight increment
in TSH secretion after 24, 48 and 72 hours (Fig. 1). Treatment of
cells with 10–9 to 10–8 mol/L of leptin caused a significant
(P£0.05) increase in TSH secretion compared to the control,
starting with 72 or 48 hours of exposition, respectively. After 72
hours, TSH secretion reached the maximum value (1.64 ± 0.01
µIU/mL/250,000 cells/72 hours) under the influence of leptin in
the concentration of 10–8 mol/L. It was significantly higher
(P£0.05) compared to the control (1.31 ± 0.06 µIU/mL/250,000
cells/72 hours) (Fig. 2). The study of the relationship between
10–10–10–8 mol/L of leptin and TSH secretion from ovine pituitary
cells in vitro showed a positive correlation (r=0.82, r=0.80, and
r=0.75 after 24, 48, and 72 hours, respectively). The addition of
leptin in the concentration of 10–7 and 10–6 mol/L decreased TSH
secretion compared to the culture with 10–8 mol/L of leptin, but at
the same time caused the elevation compared to the control.
However, the introduction of 10–5 mol/L of leptin reduced
thyroid-stimulating hormone secretion, compared with both the
control and other leptin treated cultures. The most significant
suppressive effect was observed after 72 hours of exposure of
cells to 10–5 mol/L of leptin (TSH: 1.09 ± 0.02 µIU/mL/250,000
cells/72 hours). The negative correlation between leptin in the
concentration 10–7 to 10–5 mol/L and TSH secretion from ovine
pituitary cells in vitro (r= –0.56, r= –0.81, and r= –0.88 after 24,
48, and 72 hours, respectively) was found.
Fig. 2. Changes in TSH
secretion from ovine
pituitary cells under the
influence of 10–8 mol/L
of leptin or 10–8 mol/L
of leptin and L-NAME.
a, b, c, - the mean
values obtained at the
same time (24, 48 or 72
hours) and signed with
various letters differ
significantly (P£0.05).
Fig. 3. Changes in nitric
oxide release from
ovine pituitary cells
under the influence of
10–8 mol/L of leptin or
10–8 mol/L of leptin and
L-NAME. a, b, c, - the
mean values obtained at
the same time (24, 48 or
72 hours) and signed
with various letters
differ
significantly
(P£0.05).
148
Table 1. Nitric oxide (NO) release by ovine pituitary cells in vitro under the influence of leptin after 24, 48 or 72 hours. * - significant
difference compared to the control (P£0.05).
Leptin concentration
in culture medium (mol/L)
control
10–10
10–9
10–8
10–7
10–6
10–5
NO release by ovine pituitary cells in vitro
(µmoles/mL/250 000 cells)
Time of exposition of cells to leptin (h)
24
48
72
1.4±0.08
1.7±0.11
1.9±0.11
2.2±0.16*
2.0±0.08
1.9±0.11*
1.7±0.11*
1.9±0.11*
1.9±0.08
1.8±0.11*
2.3±0.15*
2.4±0.14*
2.3±0.12*
2.4±0.14*
4.3±0.24*
2.2±0.12*
2.3±0.12*
3.8±0.22*
1.7±0.11*
2.3±0.14*
2.0±0.11
Table 2. Nitric oxide (NO) release by ovine pituitary cells in vitro under the influence of leptin and L-NAME after 24, 48 or 72 hours.
*- significant difference compared to the control (P£0.05).
NO release by ovine pituitary cells in vitro
(µmoles/mL/250 000 cells)
Leptin and L-NAME concentration
in culture medium (mol/L)
Time of exposition of cells to leptin and L-NAME (h)
24
48
72
1.4±0.09
1.7±0.11
1.9±0.12
control
1.2±0.07*
1.2±0.07*
1.7±0.11*
10–10
1.2±0.07*
1.2±0.07*
1.4±0.09*
10–9
–8
1.2±0.07*
0.9±0.05*
1.4±0.09*
10
0.7±0.04*
1.2±0.07*
1.4±0.09*
10–7
1.2±0.07*
1.4±0.09*
1.4±0.09*
10–6
1.2±0.07*
1.6±0.12
1.8±0.13
10–5
The effect of leptin on nitric oxide release by ovine pituitary
cells in vitro
The effect of leptin on NO release depended on time and its
dose. The addition of leptin in all used concentrations (10–10–10–5
mol/L) increased NO release after 24, 48 or 72 hours compared
to the control (Table 1, Fig. 3). The most stimulating effect was
exerted by 10–7 mol/L of leptin with the maximum value (4.3 ±
0.24 µM/mL/250,000 cells) after 72 hours. Therefore, leptin in
the concentration 10–10–10–8 mol/L and NO release were in
positive correlation (r=0.33, r=0.66, and r=0.97 after 24, 48, and
72 hours, respectively). Inhibition of NO synthesis by the
addition of L-NAME to the culture, irrespective of the dose of
leptin and time, annihilated this stimulating effect (Table 2, Fig.
3). It was confirmed by the negative correlation between NO
release from ovine pituitary cells and 10–10–10–8 mol/L of leptin
in the presence of L-NAME (r= –0.38, r= –0.74, and r= –0.62
after 24, 48, and 72 hours, respectively).
Changes in thyroid-stimulating hormone secretion from ovine
pituitary cells under the influence of leptin and L-NAME
Inhibition of NO synthesis by the treatment of ovine
pituitary cells with L-NAME, significantly (P£0.05) reduced
leptin-modulated secretion of TSH after 24, 48 and 72 hours
(Fig. 4). The marked (P£0.05) difference between TSH secretion
under the influence of leptin and leptin with L-NAME was
found. There was observed the significant (P£0.05) decrease in
thyroid-stimulating hormone secretion under the condition of
inhibited NO synthesis compared to the culture with leptin only
(Fig. 2). The most suppressive action was observed after 24 and
48 hours of exposure of cells to 10–5 mol/L of leptin and LNAME (TSH: 0.57 ± 0.03 µIU/mL/250,000 cells/24 hours and
0.55±0.01 µIU/mL/250,000 cells/48 hours, respectively). It was
significantly (P£0.05) lower compared to the culture with leptin
only (TSH: 1.25 ± 0.02 µIU/mL/250,000 cells/24 hours and
1.14±0.02 µIU/mL/250,000 cells/48 hours, respectively), and to
the control (TSH: 1.27 ± 0.02 µIU/mL/250,000 cells/24 hours
and 1.28±0.04 µIU/mL/250,000 cells/48 hours, respectively).
This effect was confirmed by the negative correlation between
leptin in the concentration 10–7 to 10–5 mol/L and TSH secretion
from ovine pituitary cells in vitro, modulated by leptin and LNAME (r= –0.45, r= –0.49, and r= –0.58 after 24, 48, and 72
hours, respectively). In the case of leptin and L-NAME treated
cultures, negative correlation between 10–10–10–8 mol/L of leptin
and TSH secretion (r= –0.20, r= –0.26, and r= –0.18 after 24, 48,
and 72 hours, respectively) was also found.
DISCUSSION
Leptin is an obligatory metabolic signal, which can influence
the secretion of tropic hormones e.g. TSH. However, these data
are not unequivocal and do not relate to sheep. It has been
demonstrated that leptin exerts a stimulating effect on TSH
secretion in rats. Food deprivation, and the associated low leptin
levels, resulted in decrease of TSH synthesis in the pituitary and
TRH in the hypothalamus (15, 33). In contrast, Nowak et al. (34)
noted that leptin-induced elevation in the thyroid hormones
level, probably by negative feedback, suppresses TSH release in
149
Fig. 4. Changes in TSH
secretion from ovine
pituitary cells under the
influence of leptin and
L-NAME. *- significant
difference compared to
the control (P£0.05).
female rats. However, Ortiga-Carvalho et al. (16) showed that
leptin presents a stimulating effect on the secretion of TSH in
vivo, but decreases it in vitro in male Wistar rats. The obtained
results indicated that thyroid-stimulating hormone secretion
from pituitary cells isolated from ewe lambs is dependent on
leptin concentration. It was noted that 10–10–10–6 mol/L of leptin
increases TSH secretion. The most stimulating effect of leptin on
TSH secretion was observed after cells exposure to 10–8 mol/L of
leptin. Positive correlation between 10–10–10–8 mol/L of leptin
and TSH secretion from ovine pituitary cells confirmed this
stimulating action. Whereas, the highest concentration of leptin
(10–5 mol/L) decreased TSH secretion compared to the control.
Therefore, the negative correlation between 10–7–10–5 mol/L of
leptin and TSH secretion was found. This suppressive effect may
be caused by down-regulation of the leptin receptors in pituitary
cells due to the highest leptin concentration. According to
Kosior-Korzecka (35), 10–5 mol/L of leptin significantly reduces
mRNA expression of OB-Ra and OB-Rb in ovine pituitary cells
in vitro. Moreover, Oliveira et al. (36) reported that leptin can
exert stimulating as well as inhibiting action on thyroidstimulating hormone secretion. Acute treatment of leptin
resulted in higher TSH level in rats in vivo. However, chronic
addition of leptin did not change TSH secretion. According to
our previous studies on immature seven-month old ewes, TSH
secretion in pituitary cells in vitro was also dependent on leptin
concentration. However, the most stimulating effect was
observed after the culture cells exposure to 10–7 mol/L of leptin
(unpublished data). Taking into account that leptin receptor
expression increases by around fivefold from fetal to
postpubertal life, the reason of this discrepancy may be the lower
sensitivity of pituitary cells of pubertal ewe lambs to leptin (37).
However, it is difficult to confront the results of our study with
the literature data, because no other reports on the effect of leptin
on TSH secretion by ovine pituitary cells have appeared so far.
The secretion of tropic hormones from pituitary cells can be
modulated by nitric oxide. However, the contribution of NO to
regulation of TSH secretion is not well established. It was
reported that NO may participate in the modulation of the
hypothalamic-pituitary-thyroid axis activity. It was noted that
treatment of rats with L-NAME elevates TRH levels, while
decreasing serum TSH concentration, probably by a direct effect
on anterior pituitary cells in vivo. Whereas thyroid-stimulating
hormone secretion was not changed under condition of inhibited
nitric oxide synthesis in vitro (38). According to Coiro et al.
(28), treatment with L-NAME did not modified basal release of
thyroid-stimulating hormone, but significantly reduced TRHinduced TSH secretion in humans. Therefore, it suggests that
TRH stimulates thyroid-stimulating hormone release via nitric
oxide. However, there are also contradictory reports. KasperskaZajac et al. (29) noted that in female Wistar rats NO may play an
inhibitory role in TSH secretion. The addition of L-NAME did
not change TSH concentration in plasma, while treatment with
L-arginine (the substrate for NO synthesis) resulted in decrease
in thyroid-stimulating hormone secretion. The present results
show that the leptin effect on TSH secretion in ewe lambs is
mediated by nitric oxide. Inhibition of NO synthesis with LNAME significantly reduced leptin-modulated secretion of TSH
and annihilated a stimulating effect of leptin on thyroidstimulating hormone secretion. The marked difference was
observed between TSH secretion under the influence of leptin
and leptin with L-NAME. This effect was confirmed by negative
correlation between leptin in the presence of L-NAME and TSH
secretion from ovine pituitary cells in vitro. Moreover, the
results of studies on ewe lambs pointed out that leptin in all
concentrations increases NO release in the dose-dependent way.
Leptin in the concentration 10–7 mol/L exerted the most
stimulating effect. However, the higher doses decreased NO
release from ovine pituitary cells compared to 10–7 mol/L of
leptin. Also according to other author, addition of 10–8–10–6
mol/L of leptin resulted in elevation, whereas 10–5 mol/L caused
a drop in NO release in vitro (39). Inhibition of NO synthesis
with L-NAME significantly annihilated this stimulating
150
influence. However, in our previous studies, which was carried
out on pituitary cells isolated from younger ewes (seven-month
old), TSH secretion was suppressed under the condition of
L-arginine-stimulated NO synthesis, whereas inhibition of NO
production by L-NAME caused elevation in the TSH level
(unpublished data). The explanation of these differences in the
response of pituitary cells from pubertal and mature ewes to
leptin and NO requires the further investigation. However, it is
known that regulation of TSH secretion is dependent on ovarian
steroid hormones and the phase of the oestrous cycle. There was
reported, that estrogens might stimulate thyroid-stimulating
hormone release by increasing proliferation of thyrotrophs as
well as modulate the level of receptors for the neurohormones in
thyrotropic cells (40, 41). Therefore, the reason for this
discrepancy may be the lack of the reproductive cyclicity, low
level of female steroid hormones, and then the different capacity
of thyrotropes to respond to leptin and nitric oxide in pubertal
seven-month old ewes.
The obtained results show that leptin in the doses 10–10–10–6
mol/L elevates TSH secretion from ovine pituitary cells in vitro
and this effect is mediated by nitric oxide in ten-month old
lambs. Moreover, it was observed that the highest concentration
of leptin (10–5 mol/L) decreases thyroid-stimulating hormone
secretion. It suggests that high body mass, associated with high
level of leptin can result in some disturbances in TSH release.
Taking into account that hormones of thyrotropic axis participate
in the regulation of reproductive processes in ruminants, the
consequence can be delay of puberty in ewes.
Acknowledgements: The study was supported by Polish
MSHE grant NN308598439.
Conflict of interests: None declared.
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R e c e i v e d : July 30, 2013
A c c e p t e d : January 27, 2014
Author’s address: Dr. Paulina Radwanska, Department of
Pathophysiology, Chair of Preclinical Veterinary Sciences,
Faculty of Veterinary Medicine, University of Life Sciences in
Lublin, 12 Akademicka Str., 20-033 Lublin, Poland.
E-mail: [email protected]