The pentapeptide Ipamorelin stimulates growth hormone (GH) release in pigs, sheep, dogs, rats and humans. In order to determine the effects of chronic treatment with Ipamorelin on the in vitro GH response, Ipamorelin was administered daily for 21 days to 60-day old female rats and the effects compared with those of growth hormone-releasing hormone (GHRH) (10 μg/kg body weight). Daily monitoring revealed a higher % weight gain in the Ipamorelin- pretreated group (IPG) and the GHRH-pretreated group (GPG) than in the vehicle-pretreated group (VPG). In 3-day pituitary cell monolayer cultures, basal GH release was greater in IPG and GPG than in VPG. After 3-day culture, basal GH release from individual cells was lower in IPG than in VPG and GPG. The intracellular GH content was lower in IPG and GPG than in VPG (P<0.05). In 3-day monolayer cultures, Ipamorelin (10-8 M) and GHRH (10-8 M) increased GH release in VPG and IPG, but not in GPG. Similarly, Ipamorelin (10-8 M) and GHRH (10-8 M) stimulated individual GH release in VPG and IPG. In conclusion, these data show that chronic administration of Ipamorelin to young female rats is effective in increasing body weight gain and enhancing in vitr o basal and Ipamorelin or GHRH-stimulated GH release. In the same conditions, GHRH increases body weight gain and maintains basal GH at levels similar to those produced by Ipamorelin, but without prompting any in vitro enhancement of the GH response to subsequent stimuli. These results suggest that Ipamorelin does not lead to desensitization of the GH response in young female rats, whilst GHRH produces desensitization of GH release in vitro.
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SUMMARY
The pentapeptide Ipamorelin stimulates
growth hormone (GH) release in pigs, sheep,
dogs, rats and humans. In order to determine the
effects of chronic treatment with Ipamorelin on
the in vitro GH response, Ipamorelin was
administered daily for 21 days to 60-day old
female rats and the effects compared with those
of growth hormone-releasing hormone (GHRH)
(10 μg/kg body weight). Daily monitoring
revealed a higher % weight gain in the Ipamore-
lin-pretreated group (IPG) and the GHRH-pre-
treated group (GPG) than in the vehicle-pre-
treated group (VPG). In 3-day pituitary cell
monolayer cultures, basal GH release was
greater in IPG and GPG than in VPG. After 3-day
culture, basal GH release from individual cells
was lower in IPG than in VPG and GPG. The
intracellular GH content was lower in IPG and
GPG than in VPG (P<0.05). In 3-day monolayer
cultures, Ipamorelin (10-8 M) and GHRH (10-8 M)
increased GH release in VPG and IPG, but not in
GPG. Similarly, Ipamorelin (10-8 M) and GHRH
(10-8 M) stimulated individual GH release in VPG
and IPG. In conclusion, these data show that
chronic administration of Ipamorelin to young
female rats is effective in increasing body weight
gain and enhancing in vitro basal and Ipamore-
lin or GHRH-stimulated GH release. In the same
conditions, GHRH increases body weight gain
and maintains basal GH at levels similar to those
produced by Ipamorelin, but without prompting
any in vitro enhancement of the GH response to
subsequent stimuli. These results suggest that
Ipamorelin does not lead to desensitization of
the GH response in young female rats, whilst
GHRH produces desensitization of GH release in
vitro.
Key Words: Growth hormone secretagogues –
Ipamorelin – Individual growth hormone release
in vitro – Image analysis
INTRODUCTION
Over recent years, a new series of growth
hormone (GH)-releasing peptide (GHRP) recep-
tor-active GH secretagogues (GHS) have been
developed (Raun et al., 1998); one of the proto-
types is the pentapeptide Ipamorelin, which has
been shown to be a very potent and efficacious
GH secretagogue, with no apparent collateral
effects on the release of other pituitary hor-
mones. Ipamorelin also induces longitudinal
bone growth and an increase in body weight
(Johansen et al., 1999; Svensson et al., 2000).
In 1996, a G-protein-linked receptor cloned in
the pituitary and hypothalamus was shown to be
a target of GHS (Howard et al., 1996; Pong et al.,
1996); an endogenous ligand specific to the GHS
Eur J An at, 6 (1): 37-45 (2002)
37
Chronic in vivo Ipamorelin treatment
stimulates body weight gain and
growth hormone (GH) release in vitro
in young female rats
L. Jiménez-Reina1, R. Cañete2, M.J. de la Torre 2and G. Bernal1
1- Department of Morphological Sciences, Section of Human Anatomy, School of Medicine, Córdoba, Spain
2- Department of Pediatrics, Pediatric Endocrinology Unit, School of Medicine, Córdoba, Spain
Correspondence to:
Dr. L. Jiménez-Reina. Departamento de Ciencias Morfológicas, Facultad de Medicina,Avda.
Menéndez Pidal s/n. 14004 Córdoba, Spain. Phone: 957 218259; Fax: 957 218246. E-
mail: [email protected]
Submitted: February 8, 2002
Accepted: April 16, 2002
receptor was later identified (Kojima et al.,
1999). Ipamorelin acts in a similar way to GHRP6
through a GHS-type receptor, since GHRP 6- and
Ipamorelin-induced GH release is inhibited to
similar extents by GHS receptor antagonists
(Raun et al., 1998). As in the case of other
GHRPs, chronic treatment with Ipamore l i n
seems to induce partial desensitization of the
GHS receptor (Johansen et al.,1999; Raun and
Von Voss, 1998), thus attenuating GH response.
GHS-induced GH release is due to the direct
stimulation of somatotroph cells and to indirect
stimulation through the hypothalamus, which at
least partially involves growth hormone-releas-
ing hormone (GHRH) neurons (Argente et al.,
1996; Dickson et al., 1999; Ghigo et al., 1997;
Giustina and Veldhuis, 1998; Korbonits et al.,
1999a; Korbonits et al., 1999b; Laron, 1995; Raun
et al., 1998; Smith et al., 1998). Therefore, it may
be hypothesized that if Ipamorelin reduces GH
response, this may be the result of desensitiza-
tion of the GHS receptor at pituitary and/or
hypothalamic level.
C u r rently, the potential therapeutic pro p e r t i e s
of GHRPs in patients with intact hypothalamic
function are currently being studied. These
GHRPs act directly on the hypothalamus and
pituitary, prompting GH release; they will there-
f o re not bypass the feedback loop controlling the
GH/IGF-1 axis (Peino et al., 1999; Micic et al.,
1999), inducing a more physiological pattern of
GH release. Treatment with GHRPs is well tole-
rated by patients, but re q u i res repeated adminis-
tration in order to achieve significant GH
responses (Laron, 1995; Ghigo et al., 1997;
Svensson, 1999), and in some cases this may
entail collateral effects, such as ACTH re l e a s e
(Arvat et al., 1998) or down-regulation of the GH
response (McDowell et al., 1995). Consequently,
many aspects of these chronic treatments must
be understood before this method can re p l a c e
conventional treatment with GH.
The aim of this study was twofold: 1) to
determine the effect of Ipamorelin or GHRH on
rat body weight; and 2) to determine whether
Ipamorelin or GHRH induce desensitization of
the GHS receptor at pituitary level. For this pur-
pose, we studied the effect of a 21-day treatment
with daily subcutaneous injections of Ipamorelin
or GHRH on body weight gain in young female
rats and on in vitrobasal and stimulated GH
release (overall and individual) and individual
GH content.
MATERIAL AND METHODS
Animals
Sixty-day-old female Wistar rats were used.
Animals were given free access to rat chow (IPM
R-20, Letica S.A., Hospitalet, Barcelona, Spain)
and tap water. They were housed individually
and kept under conventional conditions (tem-
perature: 22±2°C 12:12 h light/dark cycle with
lights on at 06.30) in the laboratory animal cen-
ter of the School of Medicine in Córdoba. The
rats were cared for and used in accordance with
E u ropean Council directive 86/609/ EEC
(24/11/1987).
Experiment Design
The duration of the experiment was 21 days
and the rats were weighed daily. They were
divided into three groups (n=6): the Ipamorelin-
pretreated group (IPG); the GHRH-pretreated
group (GPG); and the vehicle-pretreated group
(VPG). In the Ipamorelin-pretreated group, ani-
mals were injected subcutaneously (sc) daily at 9
am with 100 μg/kg body weight of the diacetate
salt of Ipamorelin (Novo-Nordisk, Denmark). In
the GHRH-pretreated group, animals were inject-
ed sc daily at 9 am with 10 μg/kg body weight
of GHRH1-29 (Serono Labs., Spain). The Vehicle-
pretreated group underwent the same manipula-
tion as the above groups, but received saline
serum instead of Ipamorelin or GHRH.
After the last injection, animals were decapi-
tated at 1 pm and the pituitaries were removed.
The posterior pituitaries were discarded, and the
anterior pituitaries were diced into small pieces
for cell dispersion.
Cell dispersion and cell culture
Pituitary cell cultures were pre p a red accord-
ing to Jiménez-Reina et al. (2000). Briefly, pitu-
itaries were diced into small pieces and dis-
persed in Erlenmeyer flasks with a mixture of
0.02 g/l collagenase (Type V, Sigma Chemical
Co.) and 0.01 g/l trypsin 1:250 (Sigma Chemical
Co.) in Dulbecco Modified Eagle’s Medium
(DMEM) (Sigma Chemical Co.). The Erlenmeyer
flask was placed in a humidified atmosphere of
5% CO2in air, at 37°C, for 60 minutes. The cel-
lular suspension obtained was gently pipetted
for ten minutes, centrifuged (100 g for 10 min.)
and washed in DMEM, twice. The cellular pel-
let was washed again, centrifuged, and re s u s-
pended in 10% fetal bovine serum (FBS)-
DMEM. Cell yield and viability were checked by
the trypan blue exclusion method: cell yields
were 1.64 ± 0.12 x 106/pituitary, and cell viabili-
ty was over 90% in all cell dispersions. The cel-
lular suspension was dispensed into tissue cul-
t u res consisting of 96 wells (2 x 104c e l l s /
well/200μl 10% FBS-DMEM) and incubated in a
humidified atmosphere of 5% CO2in air, at
37°C, for 3 days. Monolayer cultures were
washed twice with DMEM and fresh serum-fre e
DMEM was added. After a 4 hour incubation at
37°C and 5% CO2, the medium was decanted
and stored at -20oC .
L. Jiménez-Reina,R. Cañete,M. J. de la Torre and G. Bernal
38
GH Radioimmunoassay
GH concentrations in culture media were
m e a s u red in a double-antibody RIA using
NIDDK kits, as described previously (Jiménez-
Reina et al., 2000). All samples from each experi-
m e n t were measured in the same assay, and GH
values were expressed in ng/ml. Using this
method, the intra and inter-assay coefficients of
variation were 7% and 12% respectively.
Cell immunoblot assay (CIBA)
Pieces (1.5 x 1.5 cm) of polyvinylidene diflu-
oride transfer membrane (IMMOBILONTM Milli-
pore) were placed in multiwell plates. Three-day
monolayer cell cultures were incubated with
0.01 g/l trypsin 1:250 (Sigma Chemical Co.) 95%
air-5% CO2at 37°C for 30 minutes in order to
obtain pituitary cell suspensions, which were
placed on membranes (4x103cells/50μI DMEM)
and preincubated at 37°C in 95% air-5% CO2for
30 minutes. Then, 50 μl DMEM either alone, or
with Ipamorelin (10-8 M) or with GHRH (10-8 M),
was added and incubated for 4 hours. There-
after, the transfer membranes were fixed with
Bouin fluid for 30 minutes, and then immediate-
ly washed three times with PBS.
The standard curve and the estimated amount
of GH released from each isolated somatotroph
cell were recorded following Dobado-Berrios et
al. (1992). Briefly, various concentrations of puri-
fied rat GH (GH-RP-2, NIADK-NIH) dissolved in
bicarbonate buffer-NaCl (1μl drops), containing
between 0.78 ng and 25 ng of rat GH (increase
x2), were fixed on IMMOBILONTM membranes.
Transfer membranes were immunostained
using the extravidin-peroxidase method (EXTRA-
3, Sigma Chemical Co) modified from the
method described by Kendall and Hymer (1987).
Anti-rat GH serum (Biogenesis Ltd.) was used at
a final dilution of 1:1000. The specificity of CIBA
for rat GH was examined by removing the anti-
serum, replacing the antiserum with normal rab-
bit serum, and preadsorbing the antiserum with
rat GH (Biogenesis Ltd.) at 4°C for 24 hours. The
crossreactivity of the primary antiserum with
prolactin was also checked by incubating in the
presence of anti-rat GH IMMOBILONTM mem-
branes in which 1 μl droplets contained 25 ng/μl
of purified rat prolactin.
Image analysis
To ascertain possible changes in GH-
immunostained cell proportions in transfer mem-
brane cultures, a count of secreting and non-
secreting GH cells (Fig. 1) was performed on the
central areas of each membrane, using a gridded
eyepiece.
Image analysis was performed as described
previously (Jiménez-Reina et al., 2000; Dobado-
Berrios et al., 1992). GH-immunostained cell
blots were measured using a conventional Nikon
microscope equipped with a light source stabi-
lized at 5 volts and connected via a Hitachi tele-
vision camera to an image-analysis system con-
sisting of a computer (Pentium/Intel) with a
digital card equipped with VISILOG 4.1 software
(Noesis, Orsay, France) belonging to the Univer-
sity of Córdoba Cell Biology Department, an
additional monitor (Fujitsu, Japan), and a digital
board (KURTA). In each membrane, the halo
secretion area, the cellular area (in μm2) and
optical density of both areas (OD, in arbitrary
units) of 60 GH-immunostained cell blots were
measured. Measurements of standard intensities
define a variable that expresses the amount of
hormone immobilized per unit area or immobi-
lization density (pg/μm2) (Dobado-Berrios et al.,
1992); this enabled determination of the amount
of GH released by or theGH content (in terms of
pg/cell) of each somatotroph cell.
Statistical analysis
Results were expressed as means ± standard
errors of the means (SEM). A minimum of six
animals was used in each experimental and con-
trol group. Experiments were repeated three
times with different groups of animals. One way
repeated measures ANOVA was used to analyze
weight gain curves. The statistical significance of
i n t e r- g roup diff e rences was determined by
ANOVA, and was accepted at P<0.05. The Stu-
dent-Newman-Keuls test was used after ANOVA.
When the normalized test failed, the Kruskal-
Wallis one-way analysis of variance, followed by
the Mann-Whitney Rank Sum Test, were used.
RESULTS
Body weight
Figure 2 shows the percentage gain in body
weight (BW) with respect to the start of treat-
ment of every group. The effect of Ipamorelin
and GHRH on BW gain was immediately appa-
Chronic in vivo ipamorelin treatment stimulates body weight gain and growth hormone (GH) release in vitro in young female rats
39
Figure 1 .- Secreting (arrows) and non-secreting (arrowshead) GH
cells on transfer membrane (IMMOBILONTM Millipore)
after a 4 hour incubation by rat pituitary cells previ-
ously cultured in monolayer for 3 days. x 375.
rent and was more marked than in the vehicle
pretreated group (VPG) (p<0.05); this difference
was maintained up until day 21 (p<0.05). The
greatest gain in BW due to Ipamorelin and
GHRH (with respect saline serum) occurred dur-
ing the first week of treatment (figure 2); BW in
the second and third weeks was similar for all
three groups studied (VPG, IPG and GPG).
Monolayer culture
Cumulative basal GH release from pituitary
cells in monolayer culture after 3 days was
greater in IPG and GPG than in VPG (3720±251
and 3868±231 vs 2316±134 ng/ml; p<0.05 and
p<0.001, respectively) (Fig. 3A). No difference
was recorded for GH release between IPG and
GPG.
In the three-day monolayer cultures, basal
GH release over 4 hours in fresh medium (Fig.
3B) was similar for the three groups studied
(VPG: 141.2±12.64; IPG: 131.57±8.74; and GPG:
141.8±8.63 ng/ml) whilst Ipamorelin (10-8M) and
GHRH (10-8M) stimulated GH release in VPG
(141.2±12.6 –control– vs 189.72±18.1 –Ipamore-
lin– p<0.05; and 255.68±16.04-GHRH-p<0.001;
ng/ml) and in IPG (131.58±18.74 –control– vs
243±23.9 –Ipamorelin– and 427.1±42.8-GHRH-
p<0.001; ng/ml) (Fig. 3B). In the GPG group,
neither Ipamorelin nor GHRH stimulated GH
release (Fig. 3B).
Cell-immunoblot assay (CIBA)
After three days in monolayer culture, indi-
vidual basal GH release over 4 hours on IMMO-
BILONTM membranes was lower in IPG than in
VPG and GPG (11.2±2.58 vs 15.82±1.92 and
16.2±2.38 pg GH/cell, respectively; p<0.05) (Fig.
4A). In these conditions, Ipamorelin (10-8M)
caused an increase in individual GH release (Fig.
5) in both VPG and IPG (p<0.05), but not in
GPG. Similarly, GHRH (10-8M) stimulated indi-
vidual GH release in VPG and IPG (p<0.05), but
not in GPG (Fig. 4A).
The basal intracellular GH content in soma-
totroph cells over 4 hours in transfer membranes
was lower in IPG and GPG than in VPG
(6.05±0.76 and 5.75±0.63 v s 10.4±1.23 pg
GH/cell, respectively; p<0.05) (Fig. 4B).
Ipamorelin (10-8M) and GHRH (10-8M) only
i n c reased intracellular GH contents in IPG
(6.05±0.76 –control– vs 9.41±1.15 –Ipamorelin–
and 10.99±1.13 –GHRH-,pg GH/cell; p<0.05)
(Fig. 4B).
Individual basal GH release was heteroge-
neous. Roughly 90% of somatotrophs in VPG
released between 0 and 25 pg GH/cell, whilst
7% released more than 40 pg/cell and 10%
L. Jiménez-Reina,R. Cañete,M. J. de la Torre and G. Bernal
40
Figure 2 .- Body weight gain (%) in 60-day old female rats trea-
ted daily with Ipamorelin (100 μg/kg), GHRH (1 μg/kg)
or saline serum for 21 days. V P G: Ve h i c l e - p re t re a t e d
g roup. I P G: Ipamore l i n - p re t reated group. G P G:
GHRH-pretreated group. a: p<0.05 vs VPG.
Figure 3 .- A) Basal GH release by rat pituitary cells cultured in
monolayer for 3 days. B) GH release after a 4 hour
incubation by rat pituitary cells previously cultured in
monolayer for 3 days. VPG: Vehicle-pretreated group.
IPG: Ipamorelin-pretreated group. GPG: GHRH-pre-
treated group. a: p<0.01 and b: p<0.001 vs VPG; c:
p<0.05 vs VPG control; d: p<0.001 vs VPG control; e:
p<0.001 vs IPG control.
0-7 days
7-14 days
14-21 days
aa
VPG IPG GPG
VPG IPG GPG
VPG IPG GPG
30
20
10
0
6
4
2
0
600
400
200
0
ab
cde
e
A)
B)
Control
Ipamorelin (10-8 M)
GHRH (10-8 M)
released around 75 pg GH/cell (Fig. 6A). In IPG,
almost 14% of somatotroph cells released more
than 40 pg GH/cell (Fig. 6B). In GPG, almost
25% of somatotroph cells released between 25
and 35 pg GH /cell (Fig. 6C). With regard to
intracellular GH contents, in VPG almost 7% of
somatotroph cells contained between 30 and 40
pg GH/cell and the remainder (93%) contained
less than 20 pg GH/cell (Fig. 6D).In IPG and
GPG no somatotroph cell contained more than
20 pg GH/cell (Fig. 6E and 6F).
DISCUSSION
This study examined the effects of chronic
treatment (21 days) with Ipamorelin or GHRH on
body weight and basal and stimulated GH
release in vitro in young female rats. The results
obtained showed that both Ipamorelin and
GHRH prompted an increase in body weight and
stimulated basal GH release in vitro. GH stimu-
lation by Ipamorelin (10-8 M) and GHRH (10-8 M)
in vitro (overall and individual) was effective in
the group pretreated with saline and in the
group pretreated with Ipamorelin, but not in ani-
mals pretreated with GHRH.
Rats receiving Ipamorelin displayed signifi-
cant increases in BW with respect to the group
receiving only saline serum. An increase in BW
was also observed in rats receiving 10 μg/Kg/day
GHRH. The increase in BW was lower than that
reported in other Ipamorelin (Johansen et al.,
1 9 9 9 ) studies using the orally-administrated
derivative NN703 (Hansen et al., 1999) or G-
7039, a GH secretagogue from the Genentech
group (Clark, 1999). This was probably because
the present experiment used young female rats,
which grow faster than the adult rats used in the
above studies (Johansen et al., 1999; Raun and
Von Voss, 1998; Hansen et al., 1999). Recent
reports have suggested that the increase in BW
prompted by GH secretagogues may be due to
greater food intake, with no increase in longitu-
dinal bone growth (Lall et al., 2001). The data
obtained here show that body weight gain in
young female rats took place primarily during
the first week of treatment with Ipamorelin or
GHRH (almost 75% of total gain). We therefore
believe that weight gain would be due not to
increased food intake, in which case weight gain
would have been constant throughout treatment,
but rather to the effect of Ipamorelin on GH
secretion. No data are available to show why
body weight gain was limited to the first week of
treatment, although it is likely that the disconti-
nuous administration of Ipamorelin for 21 days,
instead of continuous infusion, served to limit
the GH response (Huhn et al., 1993).
The GH response was investigated at the end
of the study using two in vitro systems to assess
GH release in monolayer cultures and individual
GH release using the cell-immunoblot assay
method. In vivo t reatment with GHRH or
Ipamorelin increased basal GH release in mono-
layer cultures by 60% and 67% respectively with
respect to saline. It is well known that repeated
Chronic in vivo ipamorelin treatment stimulates body weight gain and growth hormone (GH) release in vitro in young female rats
41
60
40
20
0
20
10
0
a
aa
a
b
b
bb
c
eA)
B)
Control
Ipamorelin (10-8 M)
GHRH (10-8 M)
VPG IPG GPG
Figure 4 .- A) Individual GH release and B) individual GH con-
tents of transfer membrane after a 4 hour incubation
by rat pituitary cells previously cultured in monolayer
for 3 days. VPG: Ve h i c l e - p re t reated group. IPG:
Ipamorelin-pretreated group. GPG: GHRH-pretreated
group. a: p<0.05 vsVPG control; b: p<0.05 vs IPG con-
trol; c: p<0.05 vs control of VPG and GPG.
Figure 5 .- Individual GH release after Ipamorelin (10-8 M) treat-
ment on transfer membrane (IMMOBILONTM Millipore)
after a 4 hour incubation by rat pituitary cells previ-
ously cultured in monolayer for 3 days. x 375.
stimulation with GHRH prompts a decreased GH
response (Aleppo et al., 1997), although the
appearance of desensitization in the GH
response to GHRH depends more on the fre-
quency and method of administration (continu-
ous infusion vs. repeated injection) than on the
GHRH dose employed (Arsenijevic et al., 1987;
Kovacs et al., 1994; Pérez-Romero et al., 1999;
Pinski et al., 1996; Sato et al., 1994). In the pre-
sent case, repeated administration of GHRH at
24-hour intervals maintained high levels of in
vitro basal GH release, a finding similar to that
reported in vivo in prepubertal female rats
(Pérez-Romero et al., 1999) and in sheep (Pérez-
Romero et al., 2000). Bercu and Wideman (1991)
showed that chronic treatment with GHRP-6 (14
days) maintained high serum GH levels in
female rats. In the present study, similarly,
chronic administration of Ipamorelin increased
basal GH release in vitro, suggesting that, at least
in these conditions, the GHS Ipamorelin does
not elicit somatotrophic desensitization.
In vitro , s o m a t o t roph cells are able to
increase GH response after stimulus by GHRH or
Ipamorelin when obtained from animals treated
in vivowith the GHS Ipamorelin, although when
they are obtained from animals treated with
GHRH the same cells do not modify GH release.
It is therefore surprising that pretreatment with
GHRH should block the response to both
Ipamorelin and GHRH. In the case of stimulus
with GHRH, this might be accounted for by a
down-regulation of the cAMP system (Aleppo et
al., 1997), although there is no clear evidence
that this system has any major influence on intra-
cellular signalling in GHSs. Neverthless, some
research has shown that GHSs enhance cAMP
activity when adenylyl cyclase activity has been
p reviously increased; for example following
prior stimulation with GHRH (Wu et al., 1996) or
in human somatotropinomas with gsp mutations
involving intense adenylyl cyclase activity
(Adams et al., 1996). The present study did not
address the intracellular mechanisms involved in
L. Jiménez-Reina,R. Cañete,M. J. de la Torre and G. Bernal
42
0 25 50 75 0 25 50 75
Individual GH release (pg/cell)
Individual GH content (pg/cell)
0 25 50 75
0 15 30 45 0 15 30 45 0 15 30 45
60
40
20
0
A B C
80
60
40
20
0
D E F
Figure 6 .- Frequency distribution of individual GH release (A, B and C panels) and individual GH contents (D, E and F panels) of transfer
membrane after a 4 hour incubation by rat pituitary cells previously cultured in monolayer for 3 days. A, D: Vehicle-pretreated
group (VPG). B, E: Ipamorelin-pretreated group (IPG). C, F: GHRH-pretreated group (GPG).
the response to pretreatment with Ipamorelin or
GHRH, but from the data reported in the litera-
ture it may be hypothesized that there could be
some point of connection between the intracel-
lular signals of the two secretagogues that would
limits the stimulatory effect of the subsequent
stimulus.
Using the CIBA technique described by
Kendal and Hymer (1987), both the size and the
optical density of GH inmunoblot cells can be
determined, affording a parameter for individual
GH release (Jiménez-Reina et al., 2000; Dobado-
Berrios et al., 1996a; Dobado-Berrios et al.,
1996b), as opposed to the reverse hemolytic
plaque assay technique, which only permits the
measurement of plaque size. Moreover, cell-blot-
ting ensures that paracrine interactions are mini-
mized on two fronts (Ramírez et al., 1997). First-
ly, it increases the distance between individual
cells. Second, the membrane used for culture
retains a putative paracrine peptide signal in the
vicinity of the releasing cells, similar to that
reported for the cell-secreted hormone. Third,
individual GH release is heterogeneous (Frawley
and Neill, 1984) and there is a small group of
somatotroph cells that release more GH than the
rest. Consequently, some discrepancies may be
encountered between the overall GH release
data (monolayer cultures) and individual GH
release data (cell-blotting).
Although pre t r eatment with Ipamore l i n
prompted a lower individual GH release, the
secretion pattern was not very different from that
recorded in the groups pretreated with GHRH or
saline. However, there was an apparent decline
in the proportion of GH-secreting cells in the
group pretreated with Ipamorelin (data not
shown) and hence the data obtained suggest that
changes in the individual basal GH response
would be due to changes in the proportions of
GH-releasing cells, as proposed in other experi-
mental conditions following stimulation with
GHRP-6 (Goth et al., 1992). Changes in the pro-
portion of GH-secreting cells might account for
the decline in individual basal GH release in the
g roup pre t reated with Ipamorelin, where a s
cumulative basal GH release from pituitary cells
in monolayer culture increased.
Changes in the amount of hormone released
were accompanied by a decline in intracellular
GH content in the groups pre t reated with
Ipamorelin or GHRH. Indeed, chronic treatment
with GHS (GHRP-6) over 14 days (Bercu and
Weideman, 1991), or with GHRH over one week
(Stefaneanu et al., 1993), prompts a decrease in
pituitary GH contents, suggestive of somatotroph
cell activation.
Despite GH cell activation following chronic
administration of Ipamorelin, somatotroph cells
in vitroresponded to both Ipamorelin and
GHRH, individual GH release increasing. There-
fore, under the experimental conditions applied
here this response rules out any desensitization
of the GHS receptor as a reaction to Ipamorelin,
similar to that originally reported for other GHSs
(McDowell et al., 1995; Bowers et al., 1984; Clark
et al., 1989). However, in the group pre t re a t e d
with GHRH, individual GH release in vitro, stimu-
lated by Ipamorelin or GHRH, was similar to
that of controls, indicating that the somatotroph
cells were desensitizated. A similar lack of atte-
nuation of the GH response has been reported in
rats following chronic administration of a GHRH
agonist (Pinski et al., 1996). The results obtained
here fail to explain why chronic treatment with
Ipamorelin elicits stimulation of the GH response
without desensitizing the somatotroph cell. One
explanation might be that the GHS receptor
would not have received maximum stimulus,
thus reducing the negative feedback of the GH
response (Nijland et al., 1998).
In conclusion, the results obtained suggest
that in young female rats chronic treatment with
Ipamorelin or with GHRH elicits body weight
gain; pretreatment with Ipamorelin alone stimu-
lates the basal GH response in vitro; soma-
totroph cells are able to release more GH when
stimulated with Ipamorelin or GHRH. However,
pretreatment with GHRH produces a desensitiza-
tion of GH release in vitro.
ACKNOWLEDGEMENTS
The authors thank Dra. González-López for
RIA determinations and Dept. Biología Celular
(University of Córdoba, Spain) for densitometry
analysis. This work was supported in part by
Novo Nordisk, Spain. GHRH1 - 2 9 was kindly
donated by Serono Labs (Spain).
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Chronic in vivo ipamorelin treatment stimulates body weight gain and growth hormone (GH) release in vitro in young female rats
45
Miklos Góth
Charles E. Lyons
Benedict J Canny
Michael O Thorner
GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.
Pablo M. Dobado-Berrios A Ruiz-Navarro
Rafael Torronteras
Francisco Gracia-Navarro
The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.
Grazia Aleppo Stanley F. Moskal Paula A. De Grandis
Lawrence Frohman
Repeated stimulation of pituitary cell cultures with GH-releasing hormone (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desensitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cAMP. In the present study, we sought to determine if homologous down-regulation of GHRH receptor number is due to a decrease in GHRH receptor synthesis. To this end, we developed and validated a quantitative RT-PCR assay system that was capable of assessing differences in GHRH-R messenger RNA (mRNA) levels in total RNA samples obtained from rat pituitary cell cultures. Treatment of pituitary cells with GHRH, for as little as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA levels. The maximum effect was observed with 0.1 and 1 nM GHRH, which reduced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of control values, respectively (n = three separate experiments; P < 0.05). Accompanying the decline in GHRH-R mRNA levels was a rise in GH release; reaching 320 +/- 31% of control values (P < 0.01). Because of the possibility that the rise in medium GH level is the primary regulator of GHRH-R mRNA, we pretreated pituitary cultures for 4 h with GH to achieve a concentration comparable with that induced by a maximal stimulation with GHRH (8 micrograms GH/ml medium). Following pretreatment, cultures were stimulated for 15 min with GHRH and intracellular cAMP accumulation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipated, GHRH pretreatment (10 nM) significantly reduced subsequent GHRH-stimulated cAMP to 46% of untreated controls. These data suggest that GHRH, but not GH, directly reduces GHRH-R mRNA levels. To determine whether this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of adenylate cyclase. Forskolin (10 microM) significantly reduced GHRH-R mRNA concentrations (37 +/- 6% of control values) indicating that GHRH acts through the cAMP-second messenger system cascade to regulate GHRH-R mRNA. The somatostatin analogue, octreotide (10 nM), which has been previously reported to decrease adenylate cyclase activity, did not affect GHRH-R mRNA levels. Taken together, these results indicate that GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation.
Pablo M. Dobado-Berrios A Ruiz-Navarro Yolanda Almaden
Francisco Gracia-Navarro
Mammalian aging is characterized by a decline in the content and release of pituitary growth hormone (GH). However, few studies on the age-related changes in the population of GH-producing cells (somatotropes) have been carried out. We have investigated whether changes in number, ultrastructure and GH gene expression in subpopulations of somatotropes could explain the reduced GH release in aged rats. Three representative ages were studied: adult (5-month-old), old (19-month-old), and senescent (26-month-old) male rats. The total number of immunoreactive-GH cells per pituitary gland remained invariable to age. The separation of dispersed pituitary cells on a density gradient yielded two somatotrope subpopulations, of low density (LD) and high density (HD). Both subpopulations were equally represented in adults, whereas in old and senescent rats a predominance of LD-somatotropes was observed. Morphometric analysis showed that subpopulations exhibited storage and biosynthetic features inversely related. In LD-somatotropes, rough endoplasmic reticulum (RER) was more prominent but secretory granules (SG) were less abundant than in HD somatotropes. Concurrently, in situ hybridization for GH mRNA showed that GH gene expression was higher in LD-cells. Differences between subpopulations were essentially retained through the animals' lifespan, but small-sized SG, reduced RER, and low GH mRNA levels were inherent to aging both in LD- and in HD-somatotropes. The present findings demonstrate that the reduced content of pituitary GH in aged male rats is not due to a diminished number of GH-producing cells, but to the numerical predominance of scarcely granulated LD-somatotropes, combined with the decline in GH biosynthetic capacity observed in both subpopulations. In addition, age-related changes in ultrastructure and GH gene expression suggest a chronic inhibition of GH release and/or a weak stimulation of GH biosynthesis affecting both subpopulations.
Lucia Stefaneanu
Kalman Kovacs Eva Horvath J. Cronin Michael
The effect of GRH infusion on rat adenohypophysial morphology was studied by light microscopy, immunocytochemistry, in situ hybridization, and electron microscopy. Synthetic rat GRH was intravenously administered by osmotic minipumps at 14.4, 72, 360 and 720 μg/ day/rat for 1 week. In one group treated for 1 week with a daily dose of 720 μg GRH, the rats were killed 7 days after withdrawal of GRH. Control rats in which GRH was replaced by excipient, or those that received no treatment, were included as well. GRH infusion with daily doses of 360 and 720 μg resulted in a significant increase in pituitary weight and weaker GH immunoreactivity compared with other groups. Ultrastructurally, the somatotrophs were increased in size and became sparsely granulated, and the organelles involved in hormone sythesis were very prominent. The intensity of the GH mRNA signal did not differ from control animals, suggesting the desensitization of somatotrophs to GRH. The highest GRH dose induced an increased number of nuclei immunoreactive for proliferation cell nuclear antigen (PCNA). One week after GRH withdrawal, shrinkage of cytoplasm, involution of RER and Golgi complex, and a decrease of cell attachment sites indicated the reversibility of changes induced by GRH. In conclusion, GRH infusion induced, within days, hypertrophy and proliferation of somatotrophs with ultrastructural features of highly stimulated, sparsely granulated cells. Morphological changes were reversible.Endocr Pathol 4:131–139, 1993.
R G Clark Lena M S Carlsson J Trojnar I.C.A.F. Robinson
Abstract The growth hormone (GH) releasing ability of GH-releasing factor (GRF) and a GH-releasing hexapeptide, CHRP, have been studied in anaesthetized and conscious male and female rats. The GH responses to GHRP in anaesthetized rats were inconsistent, and this peptide was much less potent than GRF. Continuous iv infusions of GRF or GHRP both caused an initial GH release which was not maintained, and further GH release could be elicited by injection of GRF during an infusion of GHRP and vice versa. In contrast, conscious rats were much more sensitive to GHRP. Infusions of GHRP or GRF both caused an initial GH release. With GRF infusions, GH release continued in the normal episodic pattern whereas with GHRP infusion, GH secretion remained elevated over baseline and the normal pulsatile rhythm was disrupted. Plasma GH levels fell after stopping GHRP infusion, without an immediate resumption of normal GH pulsatility. Conscious male rats responded intermittently to injections of GRF given iv every 45 min, but when such serial injections of GRF were given during a continuous iv infusion of GHRP, the GH responses to GRF became regular and more uniform. These results suggest that GHRP prevents the normal cyclic refractoriness to GRF in male rats by disrupting cyclic somatostatin release. The greater potency of GHRP in conscious rats may also depend on the release of endogenous GRF since passive immunization with an anti-GRF serum reduced the plasma GH response to GHRP infusion. Thus in the conscious animal, GHRP may release GH by complex actions at both a hypothalamic and pituitary level.
Barry B Bercu
Carol Weideman
Richard F Walker
The purpose of this study was to compare GH secretion after the administration of GH-releasing hexapeptide (GHRP-6) in conscious male and female rats. Plasma GH was significantly elevated in female rats (six of six) compared to male rats (three of six) 15 min after administration of a single sc injection of GHRP-6 (0.5 mg/kg). In male rats, GHRP-6 administration was associated with suppression of episodic GH secretion and desensitization to a second injection administered 6 h later, whereas in female rats, GH secretion occurred after both GHRP-6 injections. After 14 consecutive days of administering GHRP-6 twice per day, mean plasma GH concentrations in males decreased from 110 +/- 91 to 2.8 +/- 0.6 ng/ml (P less than 0.05) and in females increased from 170 +/- 53 ng/ml to 361 +/- 81 ng/ml (P less than 0.05). Desensitization to GHRP-6 in conscious male rats was not observed in pentobarbital-anesthetized male rats, suggesting that GHRP-6 administration enhanced somatostatin release in the conscious state. After 14 consecutive days of GHRP-6 administration, the mean pituitary GH concentration in female rats was significantly lower than that in male rats (5.1 +/- 0.2 vs. 12.9 +/- 1.2 micrograms/mg, respectively). Lower pituitary GH concentrations in females correlated with higher GH secretion after GHRP-6 administration. Desensitization to GHRP-6 in male rats is attributed to neurohumoral factors producing their unusual pattern of episodic GH secretion, and the response is probably not typical of other species.
Yvan Arsenijevic Robert W. Rivest Aliza Eshkol
Michel Aubert
The ability of human (h)GRF-(1-29)NH2 to stimulate GH secretion was studied in cannulated adult rats. In order to suppress endogenous GRF secretion and the inhibitory action of hypothalamic somatostatin (SRIF), rats were anesthetized with sodium pentobarbital. Intravenous administration of hGRF-(1-29)NH2 elicited a dose-dependent response of plasma GH, with 250 ng/kg being the smallest effective dose in male rats. In female rats, for each dose tested (250 to 70,000 ng/kg), the GH response represented only about 60% that of male rats. Repeated iv stimulations with hGRF-(1-29)NH2 at short time intervals (45 min) produced transient desensitization of pituitary responsiveness to GRF: a blunted GH response to the second and third stimulations was observed both in male and in female rats and for each dose tested. Similar blunted responses were also obtained with repeated injections of native hGRF-(1-44)NH2. The possibility that these blunted responses could be due to incomplete suppression of hypothalamic SRIF secretion by sodium pentobarbital was excluded by the use of rats that were passively immunized against SRIF; in these rats, it was shown that at least 65% of the inhibition of the GH response after the second GRF stimulation was unrelated to SRIF action. Similar transient desensitization to repeated hGRF-(1-29)NH2 stimulations was also observed in conscious rats that were passively immunized against SRIF. This occurrence of blunted responses was shown to be related to the length of the time interval between GRF stimulations, with longer intervals resulting in less or no desensitization. It appears thus that modulation of pituitary responsiveness to the action of GRF is mediated by at least two independent mechanisms in the rat: in addition to the inhibitory action imposed by hypothalamic SRIF, which induces periods of refractoriness to the action of GRF, it was shown in this study that in the pituitary level each GRF stimulation also induces a transient desensitization of somatotrophs for about 1 h. This period of refractoriness might not be due to excessive stimulation with GRF, since it was also observed with the lowest dose of hGRF-(1-29)NH2 that gave a significant release of GH. Finally, a sex difference was confirmed for the response of anesthetized adult rats to stimulation with hGRF-(1-29)NH2, reflecting a sex steroid-induced modification of pituitary responsiveness to GRF stimulation.