The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 3886-3889
Copyright © 1998 by The Endocrine Society
The Growth Hormone Response to Hexarelin in Patients with Different Hypothalamic-Pituitary Abnormalities
Mohamed Maghnie,
Valeria Spica-Russotto,
Marco Cappa,
Michele Autelli,
Carmine Tinelli,
Patrizia Civolani,
Romano Deghenghi,
Francesca Severi and
Sandro Loche
Clinica Pediatrica (M.M., V.S., F.S.), Servizio Analisi Chimico
Cliniche (M.A.), Servizio di Biometria (C.T.), Policlinico S. Matteo,
Universitá di Pavia, Pavia; Divisione di Pediatria, Ospedale
Bambino Gesù, IRCCS (M.C.), Palidoro; Servizio di
Endocrinologia Pediatrica, Ospedale Regionale per le Microcitemie
(P.C., S.L.), Cagliari, Italy; and Europeptides (R.D.), Argenteuil,
France
Address all correspondence and requests for reprints to: Sandro Loche, M.D., Servizio di Endocrinologia Pediatrica, Ospedale Regionale per le Microcitemie, Via Jenner, 09121 Cagliari, Italy. E-mail:
sloche{at}mcweb.unica.it
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Abstract
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We evaluated the GH-releasing effect of hexarelin (Hex; 2 µg/kg, iv)
and GHRH (1 µg/kg, iv) in 18 patients (11 males and 7 females, aged
2.5–20.4 yr) with GH deficiency (GHD) whose hypothalamic pituitary
abnormalities had been previously characterized by dynamic magnetic
resonance imaging (MRI). Ten patients had isolated GHD, and 8 had
multiple pituitary hormone deficiency. All patients were receiving
appropriate hormone replacement therapy. Twenty-four prepubertal short
normal children (11 boys and 13 girls, aged 5.9–13 yr, body weight
within ±10% of ideal weight) served as controls. MRI studies revealed
an ectopic posterior pituitary at the infundibular recess in all
patients. A residual vascular component of the pituitary stalk was
visualized in 8 patients with isolated GHD (group 1), whereas MRI
showed the absence of the pituitary stalk (vascular and neural
components) in the remaining 10 patients (group 2), of whom 8 had
multiple pituitary hormone deficiency and 2 had isolated GHD. In the
short normal children, the mean peak GH response to GHRH (24.8 ±
4.4 µg/L) was significantly lower than that observed after Hex
treatment (48.1 ± 4.9 µg/L; P < 0.0001).
In the GHD patients of group 2, the mean peak GH responses to GHRH
(1.4 ± 0.3 µg/L) and Hex (0.9 ± 0.3 µg/L) were similar
and markedly low. In the patients of group 1, the GH responses to GHRH
(8.7 ± 1.3 µg/L) and Hex (7.0 ± 1.3 µg/L) were also
similar, but were significantly higher that those observed in group 2
(P < 0.0001). In the whole group of patients, a
significant correlation was found between the GH peaks after Hex and
those after GHRH (r = 0.746; P < 0.0001). In
this study we have confirmed that the integrity of the hypothalamic
pituitary connections is essential for Hex to express its full
GH-releasing activity and that Hex is able to stimulate GH secretion in
patients with GHD but with a residual vascular component of the
pituitary stalk.
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Introduction
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GH-RELEASING peptides (GHRPs) and their
nonpeptidyl analogs are a class of compounds with potent GH-releasing
activity in both animals and humans (1). Their action is mediated
through specific receptors on the hypothalamus and the pituitary (2)
and use signaling mechanisms distinct from those of GHRH and
somatostatin (3, 4). The presence of specific receptors for GHRPs
suggests that these compounds may represent synthetic analogs of an
endogenous ligand. Although their precise mechanism of action is not
completely understood, several data indicate that they may act as
functional somatostatin antagonists as well as amplifiers of GHRH
action (1).
Previous studies have shown that the GH responses to GHRP-6 and
hexarelin (Hex), a synthetic hexapeptide analog to GHRP-6, are absent
or severely blunted in patients with hypothalamic-pituitary
disconnection (5, 6), indicating that the hypothalamus is their
principal site of action, and that for GHRPs to express their full
GH-releasing activity it is essential that intact
hypothalamic-pituitary connections be present.
Magnetic resonance imaging (MRI) has been of great aid in the diagnosis
of disorders of the hypothalamic pituitary area. More recently, dynamic
MRI has allowed identification of discrete abnormalities of the
hypothalamic-pituitary stalk in patients with GH deficiency (GHD),
making it possible to visualize residual components of the pituitary
stalk with significantly more accuracy than conventional MRI (7).
Interestingly, the absence of the vascular pituitary stalk was strongly
associated with the presence of multiple pituitary hormone deficiencies
(MPHD), whereas the presence of a residual vascular component was
predictive of a less severe impairment of pituitary function (7). It
has also been shown that the GH response to GHRH was correlated with
the hypothalamic-pituitary abnormalities (8).
The aim of this study was to further investigate the site and mechanism
of action of Hex. To this end, we evaluated the GH-releasing effect of
Hex in a group of patients with GHD and MPHD whose hypothalamic
pituitary abnormalities had been previously characterized by dynamic
MRI.
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Subjects and methods
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Eighteen patients with GHD (11 males and 7 females, aged
2.5–20.4 yr) were studied. At the time of diagnosis all patients
underwent complete studies of pituitary function. In particular, GH
secretion was evaluated by arginine (0.5 g/kg, iv, given over 30 min),
insulin (0.1 U/kg, iv), and GHRH-(1–29) (1.0 µg/kg, iv) tests. GHD
was diagnosed when GH levels failed to rise above 10 µg/L after
arginine and insulin administration. The blood samples for GH
determinations were obtained at 0, 30, 60, 90, and 120 min. Thyroid
function was evaluated by determination of serum T4, free
T4 (FT4), and basal and TSH response to iv 200
µg/m2 TRH (Reflact, Hoechst, Frankfurt, Germany). Low
T4 and FT4 with low normal
basal TSH and delayed TSH response to TRH were documented in 8
patients. Pituitary-adrenal axis function, evaluated by determination
of morning ACTH and cortisol levels at 0800 h and the cortisol
response during insulin-induced hypoglycemia, were compatible with
subclinical ACTH deficiency in 8 patients. In these patients, the
cortisol response to im 0.25 mg ACTH-(1–24) (Synacthen, Ciba-Geigy,
Basel, Switzerland) was less than 350 nmol/L (normal, >550
nmol/L). Hypogonadotropic hypogonadism was diagnosed in 2 patients on
the basis of their serum FSH and LH responses to 100
µg/m2 iv GnRH (Relisorm L, Serono, Milan, Italy). Ten
patients had isolated GHD, and 8 had MPHD. All patients were receiving
appropriate hormone replacement therapy. Twenty-four prepubertal short
normal children (11 boys and 13 girls, aged 5.9–13 yr, body weight
within ±10% of ideal weight) served as controls. All control children
were referred to our institutions for short stature and were found to
have constitutional growth delay or familial short stature. Data for
these children have been previously published (10). The main clinical
and hormonal findings of the patients at the time of diagnosis are
summarized in Table 1
.
MRI studies were performed with a spin-echo technique and the use of a
1.5-T superconductive unit (Magnetom, Siemens, Iselin, NJ) before and
after gadopentetate-dimeglumine. Sagittal and coronal T1-weighted
images [400/15 (repetition time, milliseconds per echo), three
excitations, 3-mm thick sections, 256 x 256 pixels, and 20-cm
field of view] were obtained for all patients (7). MRI revealed an
ectopic posterior pituitary at the infundibular recess in all patients.
A residual vascular component of the pituitary stalk was visualized in
8 patients with isolated GHD (group 1), whereas MRI showed the absence
of the pituitary stalk (vascular and neural components) in the
remaining 10 patients (group 2), of whom 8 had MPHD and 2 had isolated
GHD. Mean serum insulin-like growth factor I concentrations was
48.5 ± 10.3 and 66.8 ± 9.0 µg/L in groups 1 and 2,
respectively (normal age- related values in our laboratory: 1–3 yr,
30–120 µg/L; 3–6 yr, 35–145 µg/L; 6–9 yr, 75–195 µg/L; 9–12
yr, 95–320 µg/L; 12–15 yr, 160–340 µg/L; 15–21 yr, 210–520
µg/L).
All subjects were tested with Hex (prepared and supplied by
Europeptides, Argenteuil, France) at a dose of 2 µg/kg, iv. Blood
samples were drawn from an indwelling catheter inserted in an
antecubital vein 15 min and immediately before injection of the peptide
and 15, 30, 45, 60, 90, and 120 min after injection. All experiments
started between 0800–0900 h after the patients fasted overnight. In
the patients, GH treatment was discontinued for 3 weeks before the test
sessions. The time elapsed between the diagnosis and the time of these
studies was 2.8 ± 1.0 yr (range, 0–11.2 yr).
GH was measured by fluoroimmunoassay using a commercial kit (AutoDELFIA
hGH, EG&G, Wallac, Oy, Finland). The intra- and interassay coefficients
of variations were 5.1% and 2.5%, respectively at 0.430 mU/L, 2.7%
and 2.1% at 5.08 mU/L, and 2.2% and 1.4% at 21.1 mU/L.
Cross-reactivity was less than 0.001% for PRL and human placental
lactogen. TSH, T3, T4,
FT4, PRL, ACTH, cortisol, and FSH/LH were
measured using commercially available RIAs. Statistical analysis of the
results was carried out using the Wilcoxon test for paired data and the
Mann-Whitney U test to compare groups; correlations between GH peaks
after GHRH and Hex administration were performed by regression
analysis. All values are given as the mean ± SEM.
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Results
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None of the subjects experienced adverse side-effects after Hex
administration. In the short normal children, the mean peak GH response
to GHRH (24.8 ± 4.4 µg/L) was significantly lower than that
observed after Hex administration (48.1 ± 4.9 µg/L;
P < 0.0001). In the GHD patients of group 2 the mean
peak GH responses to GHRH (1.4 ± 0.3 µg/L) and Hex (0.9 ±
0.3 µg/L) were similar and markedly low. In the GHD patients of group
1, the GH responses to GHRH (8.7 ± 1.3 µg/L) and Hex (7.0
± 1.3 µg/L) were also similar, but significantly higher that those
observed in group 2 (P < 0.0001). A comparison between
the GH responses to GHRH and Hex in the three groups of subjects is
shown in Fig. 1. A clear-cut difference
in the GH responses to GHRH and Hex was evident between both groups of
patients and the control group. In the whole group of patients, a
significant correlation was found between the GH peaks after Hex and
those after GHRH treatment (r = 0.746; P <
0.0001). The peak GH responses to Hex in the individual patients are
shown in Table 1
.
In the patients with isolated GHD, Hex caused significant increases in
serum ACTH (from 20.6 ± 2.1 to 32.9 ± 4.1 pg/mL;
P < 0.02) and PRL (from 7.6 ± 1.3 to 8.9 ±
1.44 ng/mL; P < 0.05) concentrations.
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Discussion
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MRI has greatly improved the diagnosis of disorders affecting the
hypothalamic-pituitary area in patients with idiopathic
hypopituitarism. More recently, fast framing MRI has provided new
information on the blood supply of the hypothalamus and the pituitary
in normal subjects and patients with pituitary diseases (10). The use
of this imaging technique has allowed identification in patients with
GHD of residual vascular components of the pituitary stalk not
recognized by conventional MRI. Interestingly, a GH response to GHRH
has been demonstrated in the great majority of GHD patients with a
residual vascular connection, as opposed to a lack of response in all
patients with unidentifiable pituitary stalk (8). In this study, we
have confirmed that Hex is unable to stimulate GH secretion in patients
with hypothalamic-pituitary disconnection, i.e. those in
whom the pituitary stalk was not visible after contrast enhancement. In
addition, we have also shown that Hex can stimulate GH secretion in
patients with residual vascular component of the pituitary stalk,
although to a lesser extent than in normal subjects. We have previously
observed a clear-cut, albeit low, GH response to Hex in some patients
in whom the pituitary stalk was not visualized by conventional MRI and
concluded that these low responses could be due to a direct somatotroph
stimulation by the peptide (5). In light of the present findings it
might be argued that the ability of Hex to elicit a GH response in
those patients could have been related to the presence of a residual
vascular connection.
A specific receptor for GHRPs has been cloned (2). Although these
receptors are found in both the hypothalamus and the pituitary, several
data suggest that the action of GHRPs is exerted mainly on the
hypothalamus. In fact, GHRPs can stimulate GH secretion by
pituitary cells in vitro (11), but their effectiveness is
far greater when the experiments are carried out on
hypothalamic-pituitary incubates (11). The effects of Hex on GH
synthesis (12) and of GHRP-6 on GH release (13) are markedly reduced in
rats and sheep after hypothalamic-pituitary disconnection. Moreover,
the GH responses to Hex and GHRP-6 are absent or severely blunted in
patients with hypothalamic-pituitary disconnection of different
etiology (Refs. 5, 6, 14 and this study). In normal subjects, the
GH responses to Hex (9) and GHRP-6 (6, 14) are consistently higher than
that elicited by a maximal dose of GHRH, whereas in adults with
intracranial lesions (9, 14), the GH response to GHRP is lower than or
similar to that observed after GHRH treatment. Also, in this study in
the patients with GHD and residual vascular connection, the mean GH
response to Hex was similar to that after GHRH, suggesting that Hex
normally may act by potentiating the action of GHRH. The enhancing
effect of GHRPs on GHRH-induced GH secretion in vivo has
been well documented (1). Interestingly, this synergistic effect is
absent in the patients with hypothalamic-pituitary disconnection (14).
In rats, passive immunization against GHRH significantly reduces Hex
stimulation of GH secretion (15). Furthermore, in animals with
transection of the pituitary stalk, the GH-releasing activity of a
nonpeptydil GH secretagogue is reduced, and it is restored to normal by
administration of GHRH (16). A recent study has also shown that
previous treatment with a GHRH antagonist eliminates most of the GH
response to GHRP-6 in man (17). Taken together, these observations
indicate that the presence of endogenous GHRH is essential for GHRPs to
express their full GH-releasing activity. Thus, although the absent GH
response to Hex observed in the patients not responsive to GHRH may
reflect either the absence of functioning somatotrophs and/or the
absence of endogenous GHRH, the observation of a sizable GH response to
both GHRH and Hex in the patients of group 1 might indicate that the
action of the latter is mediated by modulation of endogenous GHRH
action.
In the patients of this study, Hex was able to stimulate GH secretion
only in patients responsive to GHRH. Recent studies have shown that
pretreatment of neonatal rats with an anti-GHRH serum induces permanent
impairment of the somatotroph function (18), suggesting a crucial role
for GHRH in somatotroph development and function. Thus, the absence of
GH responses to either GHRH or Hex in our patients may reflect the
absence of functioning pituitary somatotrophs, a hypothesis put forward
by other investigators (14). Alternatively, even a normal number of
pituitary somatotrophs chronically deprived by endogenous GHRH may need
to be primed to normally synthesize and release GH (19).
In conclusion, we have confirmed that the integrity of the hypothalamic
pituitary connections is essential for Hex to express its full
GH-releasing activity and have shown that Hex is able to stimulate GH
secretion in patients with GHD but with a residual vascular component
of the pituitary stalk.
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Acknowledgments
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Helpful discussion with Prof. Eugenio E. Müller is greatly
acknowledged.
Received April 22, 1998.
Revised July 7, 1998.
Accepted July 14, 1998.
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Top
Abstract
Introduction
; 1 µg/kg, iv) and Hex (
; 2 µg/kg, iv) in 24 short
normal children (A), the patients with GHD in group 1 (B), and the
patients with GHD in group 2 (C).