Am J Physiol Endocrinol Metab 282: E943–E951, 2002.
First published January 8, 2002; 10.1152/ajpendo.00537.2001.
Pulsatile growth hormone secretion persists in genetic
growth hormone-releasing hormone resistance
HIRALAL G. MAHESHWARI,1 SUZAN S. PEZZOLI,2 ASAD RAHIM,3 STEPHEN M. SHALET,3
MICHAEL O. THORNER,2 AND GERHARD BAUMANN1
1
Center for Endocrinology, Metabolism and Molecular Medicine, Department of Medicine,
Northwestern University Medical School, and Veterans Administration Chicago Health System,
Lakeside Division, Chicago, Illinois 60611; 2Department of Internal Medicine, University of Virginia
Health System, Charlottesville, Virginia 22908; and 3Department of Endocrinology,
Christie Hospital, Withington, Manchester M20 4BX, United Kingdom
Maheshwari, Hiralal G., Suzan S. Pezzoli, Asad
Rahim, Stephen M. Shalet, Michael O. Thorner, and
Gerhard Baumann. Pulsatile growth hormone secretion persists in genetic growth hormone-releasing hormone resistance.
Am J Physiol Endocrinol Metab 282: E943–E951, 2002. First
published January 8, 2002; 10.1152/ajpendo.00537.2001.—
Growth hormone (GH) secretion is regulated by GH-releasing
hormone (GHRH), somatostatin, and possibly ghrelin, but uncertainty remains about the relative contributions of these
hypophysiotropic factors to GH pulsatility. Patients with genetic GHRH receptor (GHRH-R) deficiency present an opportunity to examine GH secretory dynamics in the selective absence
of GHRH input. We studied circadian GH profiles in four young
men homozygous for a null mutation in the GHRH-R gene by
use of an ultrasensitive GH assay. Residual GH secretion was
pulsatile, with normal pulse frequency, but severely reduced
amplitude (⬍1% normal) and greater than normal process disorder (as assessed by approximate entropy). Nocturnal GH
secretion, both basal and pulsatile, was enhanced compared
with daytime. We conclude that rhythmic GH secretion persists
in an amplitude-miniaturized version in the absence of a
GHRH-R signal. The nocturnal enhancement of GH secretion is
likely mediated by decreased somatostatin tone. Pulsatility of
residual GH secretion may be caused by oscillations in somatostatin and/or ghrelin; it may also reflect intrinsic oscillations
in somatotropes.
growth hormone-releasing hormone receptor; somatostatin;
pituitary gland; short stature
GROWTH HORMONE (GH) IS SECRETED
in a pulsatile or episodic manner. This secretion pattern is principally
governed by hypothalamic growth hormone-releasing
hormone (GHRH) and somatostatin (see Ref. 23 for
review). Ghrelin (39) and/or a similar endogenous ligand for the GH secretagogue (GHS) receptor may also
participate in GH pulse generation, although its role in
physiological GH secretion is still largely unknown.
GHRH and ghrelin stimulate pituitary GH release,
whereas somatostatin inhibits it. In addition to its
acute GH-releasing action, GHRH also stimulates GH
Address for reprint requests and other correspondence: G. Baumann, Northwestern Univ. Medical School, 303 East Chicago Ave.,
Chicago, IL 60611, (E-mail: [email protected]).
http://www.ajpendo.org
synthesis (3) and somatotrope proliferation during pituitary ontogeny (41, 68).
The pulsatile pattern of GH secretion and its relation
to GHRH and somatostatin rhythms has been evaluated extensively in a number of species (23), and a
concept of GHRH-induced GH pulses, modulated by
prevailing somatostatin tone and rapid somatostatin
oscillations, has been formulated (65). A role of ghrelin
in this interplay can be postulated but remains unproved. This model is supported by studies using immunoneutralization of GHRH and somatostatin (65,
80), GHRH, or somatostatin antagonists (5, 33, 34, 73),
as well as direct pituitary portal sampling in experimental animals (18, 20, 55, 66). A reciprocal relationship between GHRH and somatostatin, where GHRH
pulses coincide with somatostatin troughs, has been
reported in rats (37, 55, 65), but this pattern is less
clear in sheep or pigs, where more complex relationships between oscillations in GHRH, somatostatin, and
GH prevail (8, 15, 20, 66). Thus important species as
well as sex differences exist in the regulation of GH
secretion. Additional complexities are imposed by nutritional status, sex steroid milieu, and feedback by
insulin-like growth factor (IGF) I and GH. In humans,
where hypophysial-portal sampling is not feasible, the
roles of GHRH and somatostatin have been inferred
from studies with continuous, presumably saturating
infusions of GHRH, GHS, and somatostatin or its analogs (7, 13, 32, 35, 61, 72, 79) as well as by the use of
a GHRH antagonist (33, 34). These studies have provided evidence for a predominant role of GHRH and an
additive role of somatostatin in GH pattern regulation
in humans. However, despite many years of ingenious
investigations, the precise relative contributions of the
hypophysiotropic factors to minute-to-minute GH secretion remain difficult to dissect in the intact organism.
One strategy used to gain a better understanding is
to selectively remove one factor or its input from the
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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Received 3 December 2001; accepted in final form 31 December 2001
E944
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
SUBJECTS AND METHODS
Four adult males (patients 4, 5, 23, and 35 in Ref. 46) with
a homozygous GHRH-R defect, aged 23, 28, 28, and 30 yr,
respectively, participated in the study. Their genetic, physical, and endocrine characteristics have been described in
detail previously (4, 45, 46, 49). None had ever been treated
with GH or any other form of endocrine therapy. They traveled from Pakistan to Chicago and were admitted to the
Northwestern University General Clinical Research Center
(GCRC). After acclimatization for 3 days, the studies were
initiated. The study protocol was approved by the Northwestern University Institutional Review Board, and the patients
gave informed consent. An intravenous cannula was placed
in a forearm vein at 7 AM, and starting at 8 AM, blood (1 ml)
was drawn every 10 min over a 24-h period. Blood samples
were immediately added to EDTA-containing 2-ml Vacutainer tubes, cooled on ice, and centrifuged within 30 min.
The volume of the EDTA solution in the Vacutainers was
measured as 36.2 ⫾ 0.28 ␮l (mean ⫾ SE; n ⫽ 9). Plasma was
kept frozen until assayed. The patients remained ambulatory
at the GCRC, ate their meals (Pakistani food) at regular
times, and were allowed to sleep without disturbance during
the night. All patient activities were recorded by the nurses
on a continuing basis. Blood sampling during sleep was
performed from outside the room via long tubing. Blood
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contained in the dead space of the tubing was reinjected. All
patients tolerated the procedure well, and no adverse effects
were observed.
GH was measured at the University of Virginia by a
sensitive chemiluminescence assay (9) with a limit of detection of 0.01 ␮g/l and a coefficient of variation ranging from 3.9
to 8.8% in the measurement range relevant for this study. All
samples derived from the four 24-h profiles were measured in
a single assay to minimize variability due to technical reasons. No correction was made for the plasma dilution (⬃3%)
by the EDTA solution derived from the Vacutainers.
To assess a potential contribution of ghrelin (or a similar
endogenous GHS receptor ligand) to fluctuations in plasma
GH levels, we also reassayed GH levels by chemiluminescence assay in plasma specimens obtained during a previous
study (45) that had examined the effect of hexarelin in the
same four patients. Those samples had been stored at ⫺20°C
since the original assay.
To determine the degree to which intrinsic assay variability (technical variation) contributes to apparent pulsatility,
we assayed a plasma pool (GH level 0.038 ␮g/l) 136 times in
duplicate, arranged the results into a pseudo-time series akin
to a circadian profile, and subjected the pseudo-series to the
statistical tests used for the circadian profiles. Technical
oscillations in this pseudo-series were found to be random
and were limited to ⱕ0.01 ␮g/l. The pseudo-series also
showed an absence of trends, such as assay drift, and absence
of features characteristic of hormone secretion such as progressive increases/decreases in sequential samples.
Statistical analysis of the 24-h profiles was performed by
cluster analysis, an objective pulse detection program (74),
deconvolution analysis, a modeling program designed to dissect various aspects of hormonal time series such as secretion
rate and clearance (75, 76), and the approximate entropy
(ApEn) statistic, an estimate of underlying process irregularity (52, 54) [deconvolution and ApEn (1, 20% SD) analyses
were kindly performed by J. Y. Weltman and Dr. S. M.
Pincus, respectively]. The deconvolution method was used to
derive pituitary secretion parameters and GH half-life.
Pulses with amplitudes of ⱕ0.01 ␮g/l (and derived parameters) were ignored, because they have been shown to represent primarily technical variation (12) (for exceptions, see
legend to Fig. 1). Minor pulsations within a larger pulse were
considered to be part of that composite pulse (28). Differences
between daytime and nighttime GH secretion were assessed
by comparing 1) mean overall GH levels and 2) mean nadir
GH levels between the first and last 12 h of the sampling
period (8 AM-8 PM vs. 8 PM-8 AM), or between 8 AM and
sleep onset and sleep onset and 8 AM. Areas under the curve
in GH stimulation tests were determined by the trapezoidal
rule. Summary data are expressed as means ⫾ SD. Comparisons between patient and normative data were made by
t-test or by the Mann-Whitney ranked sum test where data
were not normally distributed or variances were unequal.
RESULTS
The four circadian plasma GH profiles are shown in
Fig. 1A. All four patients had extremely low overall GH
levels compared with normal subjects. Their 24-h mean
GH levels were 0.052, 0.020, 0.021, and 0.046 ␮g/l,
respectively (Table 1). This was consistent with their
phenotype of isolated GH deficiency and their lack of
response to several provocative stimuli for GH. However, it is also apparent that plasma GH levels fluctuated in a manner qualitatively similar to that in nor-
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system. This has been partially achieved by immunoneutralization studies (65, 80) or treatment with antagonists against GHRH (33, 34, 73) or somatostatin
(5). However, pharmacological approaches often have
limitations because of incomplete efficacy or lack of
complete specificity. Genetic approaches frequently
yield more definitive results because of their greater
specificity. The recent identification of genetic GHRH
receptor (GHRH-R) deficiency in humans presents an
opportunity to examine GH secretion in the selective
and complete absence of GHRH input.
We (4, 46) and others (50, 78) recently reported a
syndrome of GH-deficient dwarfism resulting from a
nonsense mutation in the GHRH-R gene (dwarfism of
Sindh). This null mutation is predicted to truncate the
GHRH-R near its amino terminus. In its homozygous
state, the mutation causes severe, isolated GH deficiency with inability to respond to GHRH and several
other GH provocative stimuli, including hexarelin (4,
45, 46, 50, 78). Affected patients also have pituitary
hypoplasia, presumably due to lack of normal somatotrope development (49, 50). Other inactivating mutations in the GHRH-R gene exhibit a very similar phenotype (29, 31, 59, 60, 64). The murine homolog is the
little (lit/lit) mouse, which harbors an inactivating missense mutation resulting in GH deficiency (14, 16, 24,
41). In all of these cases, GH production is inadequate
to sustain normal somatic growth, thereby proving the
crucial nature of GHRH for a functioning GH-IGF axis.
However, little is known about the degree of residual
GH secretion and its temporal pattern. We have employed this unique model of complete GHRH resistance
at the GHRH-R to examine residual GH secretion in
the absence of GHRH-R input, thereby gaining new
insight into the roles of somatostatin and perhaps
ghrelin (or its analogs) in generating pulsatile GH
secretion.
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
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Fig. 1. Diurnal growth hormone (GH) secretion in patients with GH-releasing hormone receptor (GHRH-R)
deficiency. A: plasma GH concentration profiles (10-min sampling intervals). Note the low numbers on the ordinates
and their different scales. B: GH secretion profiles, derived from plasma GH concentration profiles by deconvolution. Horizontal bars denote observed sleep periods. The GH secretion profile for patient 2 is drawn as a dashed line
because of the marginal reliability of deconvolution at these low secretion levels (see text). We did not derive
numerical deconvolution parameters in Table 1 for that reason. Nevertheless, the GH concentration profile of
patient 2 is visually suggestive of secretory episodes and enhanced GH secretion at night, and both the approximate
entropy (ApEn) values and night-day comparison of overall and nadir GH levels show highly significant differences
from a random oscillatory profile (see text and Table 1). It is for this reason that we show the secretion profile as
a tentative graph for the reader’s consideration. In this case, we waived the ⱕ0.01 ␮g/l increment/decrement
criterion for designating a peak and let the deconvolution program decide what constitutes a secretory episode. It
should be noted that the deconvolution program did not identify any pulses in the pseudo-time series derived from
the replicate assay of a single plasma sample, even when increment/decrement criteria were set to zero.
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E946
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
Table 1. GH secretion characteristics, as derived from deconvolution and ApEn analyses
Patient
24-h Mean
GH (␮g/l)
Peak Frequency,
no./24 h
Mass Secreted/
Burst, ␮g/l
1
0.052
18
0.081
2
0.020
—
—
3
0.021
16
0.016
4
0.046
12
0.042
Mean ⫾ SD
0.035 ⫾ 0.02
15.3 ⫾ 3.1
0.046 ⫾ 0.033
Normal young men (mean ⫾ SD)
Thorner*
1.39 ⫾ 0.77
19.0 ⫾ 4.7
3.82 ⫾ 1.99
Ref. 28
1.78 ⫾ 0.79
12.0 ⫾ 1.2
7.13 ⫾ 2.31
P values patients vs. Thorner et al. (Ref. 53 for ApEn)
P ⫽ 0.005
0.231
0.014
GH Production
Rate
Basal GH Secretion
Rate
Peak HalfDuration, min
t1/2, min
27.1
—
26.1
30.3
27.8 ⫾ 2.2
14.6
—
16.1
15.7
15.5 ⫾ 0.8
3.41
—
1.42
2.95
2.59 ⫾ 1.04
1.95
—
1.17
2.44
1.85 ⫾ 0.64
1.006
1.708
1.133
1.287
1.284 ⫾ 0.305
26.8 ⫾ 6.7
24.8 ⫾ 8.3
16.5 ⫾ 4.2
17.3 ⫾ 5.9
73.3 ⫾ 36.1
80.2 ⫾ 37.9
3.34 ⫾ 2.81
ND
0.240 ⫾ 0.115†
ND
0.797
1.00
0.007
0.275
1.6 ⫻ 10⫺5
␮g 䡠 l⫺1 䡠 24 h⫺1
ApEn
mal subjects. This included peaks occurring at 1- to 2-h
intervals, higher overall levels at night, a major nocturnal secretory pulse, and the composite nature of
secretory episodes consisting of sequential “volleys.”
Pulses were detected by both the cluster and the deconvolution programs with a high degree (89.5%) of
concordance. There was no apparent relation of GH
pulses to eating or any other recorded activity (ambulation, talking to staff, watching television, etc.). In all
four patients, nocturnal overall plasma GH levels were
significantly higher (P ⬍ 0.001) than daytime GH levels regardless of whether nocturnal was defined as
commencing at 8 PM or at sleep onset. Similarly, in all
patients, the nocturnal nadir GH levels were significantly higher (P ⬍ 0.001) than those during the day,
with the use of either of the two definitions for nocturnal. In contrast to the patients’ circadian profiles, neither cluster nor deconvolution analysis detected any
pulses in the pseudo-time series derived from the replicate assay of a single plasma sample.
Figure 1B depicts the deconvoluted GH secretion
profiles. The higher secretory activity during the night
is apparent. Two patients showed no detectable GH
secretion during the daytime, and in one (patient 2),
even the nocturnal secretion was so low that reliable
quantification was questionable. We omitted those data/time periods from the overall analysis. Because of
this limitation, pulse frequency data in three of the
four patients are based on partial circadian (primarily
nocturnal) profiles.
Table 1 summarizes the numerical data descriptive
of these circadian profiles and the deconvolution and
ApEn results. In one patient (patient 2), plasma GH
values were near the detection limit throughout the
day, a fact that rendered deconvolution difficult. In
another (patient 3), levels were similarly low during
the daytime, imposing the same limitation for the first
half of the diurnal profile. Nevertheless, the remaining
information showed that there are peaks occurring on
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average every 94 min, similar (P ⫽ 0.231) to that in
normal young (20–28 yr old) male subjects (every 76
min) when examined by the same ultrasensitive assay
(M. O. Thorner, unpublished data) or in middle-aged
(47 ⫾ 3.5 yr old) males (every 72 min) when an equally
sensitive immunofluorometric assay (detection limit
0.0115 ␮g/l) was used (71). GH plasma half-life (15.5 ⫾
0.8 min) was also similar to that in normal subjects (17,
28). The GH production per pulse was 45- to 450-fold
lower than normal, and the daily GH production rates
were 1.8–6.2% of the average GH production rate for
normal males in the same age group (28). Basal (i.e.,
apulsatile) secretion accounted for a high proportion
(57–83%) of the total GH secretion, in contrast to
normal men, where ⬃95% of daily GH production is
derived from secretory pulses (71). It should be recognized that the numerical values listed here and in
Table 1 are based on only three deconvolution profiles
and are partly derived from GH measurements near
the assay detection limit. They should, therefore, be
considered approximations.
ApEn values were intermediate between zero and
maximal (⬃1.844 for this data series length), implying
GH dynamics that were neither flat nor random (patient 2 approached maximal ApEn values, implying a
high degree of randomicity). Furthermore, ApEn values were higher in all four subjects than in normal
males, indicating a greater degree of disorderliness, or
lack of quiescence between peaks.
The observed pulsatility in the absence of a GHRH
signal raises the question of what drives this pulsatility. To gain information about the potential involvement of ghrelin or a related factor, we reexamined by
ultrasensitive chemiluminescence assay the response
of these same patients to hexarelin, previously determined only by conventional GH assay (45). All four
patients showed some, albeit very minor, GH responses
to hexarelin (Fig. 2). This finding is consistent with the
responses to GH-releasing peptide-2 reported for pa-
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GH, growth hormone; ApEn, approximate entropy; ND, not determined. Deconvolution data on patient 2 are not shown because his GH
secretion rate was too low for reliable determination (see text). GH production rate includes both basal and pulsatile components. * Thorner
(unpublished data) in 11 normal, lean males, age 20–28 yr, body mass index (BMI) 21.1–26.4 kg/m2, with the identical GH assay and 10-min
sampling intervals represents the best normative match to the present study in terms of subject characteristics and methodology. Hartman
et al. (28) reported data in 12 lean males, age 22–28 yr, BMI 21–29 kg/m2, with a less sensitive immunoradiometric assay and 10-min
sampling intervals. Pulse frequency is in part dependent on assay sensitivity, because pulses below the detection limit of the assay go
undetected. For this reason, frequencies tend to be greater with more sensitive assays. For example, van den Berg et al. (71) reported 19.9 ⫾
4.5 peaks/24 h in middle-aged men by use of an immunofluorometric assay with a sensitivity of 0.0115 ␮g/l.
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
tients affected by other inactivating mutations in the
GHRH-R gene (25, 58). The responses, based on area
under the curve, were 0.03–0.73% of what is typically
seen in normal subjects (2, 22). There was a significant
correlation (r ⫽ 0.973, P ⫽ 0.027) between mean
plasma GH in a given patient and his response to
hexarelin (area under the curve). There was no correlation between pituitary volume (49) and either mean
plasma GH or hexarelin response (P ⬃ 0.4).
DISCUSSION
Fig. 2. Plasma GH responses to hexarelin (2 ␮g/kg iv) in 4 patients
with GHRH-R deficiency, as measured by sensitive chemiluminescence assay. Note that the scale on the ordinate extends only to 1.5
␮g/l. The numbers next to the curves refer to patient numbers listed
in Table 1.
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GH pulsatility, such as pulse frequency, half-duration
of peak width, and plasma half-life, were similar to
those in normal subjects (28, 71). ApEn analysis confirmed that GH dynamics in these patients are both
real and nonrandom and that there is a greater degree
of process disorder than in normal men. Our findings
are in substantial agreement with another report on
GH secretion in two patients with a different inactivating mutation in the GHRH-R gene (58).
The possibility must be considered that residual GH
secretion was due to incomplete resistance to GHRH,
either because low levels of GHRH-Rs were present or
through interaction of GHRH with other, related receptors [e.g., vasoactive intestinal peptide (VIP) and/or pituitary adenylyl cyclase-activating polypeptide (PACAP)
receptors]. Theoretically, GHRH-R expression, despite
the mutation, could result from an alternative translational start site downstream from the mutation or from
low-level readthrough through the stop codon. Scrutiny
of the entire GHRH-R gene (Ref. 44 and GenBank accession no. AC005155) reveals no downstream start site
capable of encoding a functional GHRH-R [this includes
reported splice variants (57)]. Furthermore, on the basis
of the strength of the mutant stop codon and its RNA
context as a chain terminator, as well as information
about expression levels of mutant proteins resulting from
similar nonsense mutations in other human diseases, we
consider it highly unlikely that readthrough occurs at a
level sufficient to explain our findings. GHRH binds with
low affinity to VIP/PACAP receptors (26, 40, 77), and
activation of type I PACAP receptors occurs at GHRH
concentrations of 10–100 nM, levels at least 100-fold
higher than those needed for GHRH-R activation (21).
Because the GHRH concentration in the hypophysialportal blood of our patients is unknown, it is impossible to
ascertain directly whether residual GHRH action
through PACAP/VIP receptors is responsible for GH pulsatility. It is reasonable to postulate that, in GHRH-R
deficiency, portal GHRH levels are increased because of a
lack of GH and IGF-I feedback on hypothalamic GHRH
neurons. However, hypothalamic GHRH expression in
the little mouse is only about threefold above normal (19);
hence, the magnitude of the postulated GHRH increase
in portal blood would appear to be modest. Furthermore,
Roelfsma et al. (58) found no GH response to a pharmacological dose of GHRH in two patients with GHRH-R
deficiency, although those same patients were able to
respond to GH-releasing peptide-2. In the aggregate,
these observations do not support a significant role for
GHRH in GH pulse generation in our patients. Thus,
whereas we cannot formally exclude residual GHRH action mediated through alternate receptors, we consider
this a remote possibility.
It is generally believed that, in humans, GH secretory spikes result primarily from GHRH pulses and
that their amplitude, timing, and interpulse nadirs are
modulated by somatostatin (see introductory section).
Whether or not ghrelin plays a role in pulsatility is not
yet known. Oscillations in somatostatin may also induce GH pulses such as those seen in the presence of
constant, high levels of GHRH (72, 79) or GHS (32, 35).
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The present study was designed to evaluate GH
secretion patterns in vivo in the selective absence of
GHRH input to the somatotrope, secondary to genetic
GHRH-R deficiency. The study demonstrates that, under those circumstances, some pulsatile GH secretion
persists, albeit at greatly decreased amplitudes. Also
preserved is another feature of GH secretion, namely
enhanced GH secretion during the night, with a dominant sleep-related GH pulse. Rhythmicity of GH secretion dynamics is not only visually apparent but was
also detected by three objective, independent methods:
cluster, deconvolution, and ApEn. Without GHRH input, the amplitude of GH secretory spikes is two to
three orders of magnitude lower than normal, resulting
in severe clinical and biochemical GH deficiency (1, 4,
29, 46, 50, 78). The sensitive GH assay employed permitted robust GH measurements in two patients
throughout the day and in an additional patient at
night (100% of the GH values exceeded two times the
minimal detectable concentration in patients 1 and 4,
and 100% of nocturnal values exceeded twice the detection limit in patient 3). Of interest, the four patients
we studied varied over a threefold range in their GH
production and peak amplitude despite the fact that
they carried the same genetic defect. However, there
was no correlation between GH production and statural height or serum IGF-I level. Other parameters of
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GH SECRETION IN GHRH RECEPTOR DEFICIENCY
AJP-Endocrinol Metab • VOL
neurons to release GHRH (47); direct pituitary effects
are modest and seen only at pharmacological levels (42,
43). Thus none of these neuropeptides appears to be a
compelling candidate for driving residual GH dynamics in our patients.
Pulsatility of GH secretion has been observed, albeit
at varying amplitudes, under most, if not all, experimental conditions, including infusions with GHRH (72,
79), GHS (27, 32, 35), and somatostatinergic agents (7,
13, 61) alone or in combination (61), GHRH antagonist
treatment (34), and now, genetic GHRH resistance.
Thus, pulsatility appears as a very robust feature of
GH secretion that persists despite attempts to disrupt
hypothalamic input. This phenomenon suggests either
that there is redundancy among hypothalamic factors
in driving pulsatility or that a fundamental secretory
rhythm is inherent in the somatotrope. Indeed, both
possibilities may be true. The coordinate GHRH-somatostatin rhythm in the rat is an example of the former.
Rat somatotropes incubated in vitro without hypothalamic peptides secrete GH in an oscillatory manner, a pattern that is related to oscillating transmembrane calcium fluxes (30). The amplitude, but
not the frequency, of these oscillations is modulated
by GHRH and somatostatin (67). Furthermore, monkey hemipituitary explants in a perifusion chamber
secrete GH in a pulsatile pattern (63). These are
examples of episodic secretory activity intrinsic to
the somatotrope. These in vitro oscillations occur
with a higher frequency (2–13/min and 12–15/h,
respectively) than what is observed in vivo. However,
the relatively short observation times of these studies (5 and 75–150 min, respectively) preclude conclusions about the potential existence of longer periodicities. Thus, it is unclear how much intrinsic
somatotrope pulsatility contributes to the residual
GH pulse pattern observed in the present study.
In summary, we report that patients with genetic,
complete GHRH resistance exhibit residual pulsatile
GH secretion with a greatly diminished amplitude yet
normal frequency. The diurnal profile is qualitatively
similar to normal, with enhanced GH secretion at
night, including a raised baseline and higher peaks.
These findings confirm the critical need for GHRH as a
principal pacesetter and amplifier of GH secretion but
also illustrate that other factors contribute to the pulsatile and diurnal pattern of GH secretion in men. We
speculate that lowered somatostatin tone and rhythmic withdrawal are likely responsible for the nocturnally enhanced GH secretion, but we cannot exclude a
role for ghrelin or a similar endogenous GHS in pulse
generation. The intrinsically oscillatory nature of GH
secretion by somatotropes in the absence of hypothalamic input may also contribute to the observed pulsatility.
We thank Judy Y. Weltman for assistance with the deconvolution
analysis and Dr. Steven M. Pincus for help with the ApEn analysis
and interpretation.
This work was supported by National Institutes of Health (NIH)
Grants RR-00048, RR-00847 (General Clinical Research Centers of
Northwestern University and University of Virginia, respectively), a
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The paramount importance of GHRH for GH pulses of
normal amplitude is obvious from the present study, in
agreement with previous studies with a GHRH antagonist. Administration of the competitive GHRH antagonist (N-Ac-Tyr1,D-Arg2)-GHRH-(1–29) largely suppressed spontaneous GH secretion as well as the
response to provocative pharmacological stimuli (33,
34, 51). However, in those studies, GH secretion was
not completely abolished, a finding that was attributed
to incomplete pharmacological blockade. The present
study shows that, in men, the fundamental GH rhythm
persists in a miniaturized version even in the absence
of a GHRH-R signal. Therefore, the incomplete abolition of GH secretion by GHRH antagonist treatment
may in part be explained by this GHRH-independent
GH rhythm. Ours is a more severe paradigm of GHRH
resistance than is GHRH antagonist treatment of normal subjects because of its chronicity, somatotrope
hypoplasia, and impact on GH synthesis. GHRH can
therefore be viewed to play a dual role in GH secretion.
First, it acts as a pacesetter for GH secretory episodes
through its own pulsatile pattern in the pituitary portal system. This has been directly shown in sheep,
where there is a high concordance between hypophysial-portal GHRH pulses and GH pulses in the peripheral circulation (8, 20). It appears likely that the same
obtains in humans, although no direct information is
available. Second, GHRH acts as an important amplifier of an underlying GH rhythm, as revealed in the
present study. Among the three biological actions of
GHRH, somatotrope proliferation (6), GH synthesis
(3), and GH release (11), it is primarily the first and
second that subserve this amplifier role.
The present study also indicates that factors other
than GHRH contribute to the augmented nocturnal
GH secretion. Both baseline and pulse amplitudes
were higher at night than during the day in our patients, a phenomenon that clearly cannot be attributed
to GHRH. This is in agreement with other, more circumstantial evidence that enhanced nocturnal GH secretion is not fully attributable to GHRH (36, 48, 62,
69) and supports the concept that decreased somatostatin tone contributes to enhanced nocturnal GH
secretion (70). In the absence of GHRH action, it is
either somatostatin withdrawal, ghrelin pulses, or another, unknown oscillator that must be responsible for
residual nocturnal pulse generation. The magnitude of
the GH response to hexarelin in our patients is comparable to the spontaneous pulse amplitudes observed;
hence, ghrelin would be a theoretical possibility. However, in the absence of any information about the
physiological role of ghrelin in GH secretion, somatostatin would appear the most likely candidate at
present. Other neuropeptides to be considered here are
PACAP, VIP, galanin, and perhaps other hypophysiotropic factors acting through their own receptors.
PACAP can act as a GH secretagogue in the rat but
appears to lack this property in humans (see Ref. 56 for
review). Similarly, VIP does not stimulate GH release
in normal humans (10, 38). Galanin affects GH secretion primarily at the hypothalamic level by acting on
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
travel grant from Pharmacia & Upjohn, grants from the National
Science Foundation, the Department of Veterans Affairs, and the
Northwestern Memorial Foundation (G. Baumann), and National
Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-32632 (M. O. Thorner).
This study was presented in part at the 82nd Meeting of the
Endocrine Society, Toronto, Canada, June 2000, and reported in
abstract form (Program 82nd Meeting of the Endocrine Society,
p. 483).
REFERENCES
AJP-Endocrinol Metab • VOL
14. Donahue LR and Beamer WG. Growth hormone deficiency in
‘little’ mice results in aberrant body composition, reduced insulin-like growth factor-I and insulin-like growth factor-binding
protein-3 (IGFBP-3), but does not affect IGFBP-2, -1 or -4.
J Endocrinol 136: 91–104, 1993.
15. Drisko JE, Faidley TD, Chang CH, Zhang D, Nicolich S,
Hora DF, McNamara L, Rickes E, Abribat T, Smith RG,
and Hickey GJ. Hypophyseal-portal concentrations of growth
hormone-releasing factor and somatostatin in conscious pigs:
relationship to production of spontaneous growth hormone
pulses. Proc Soc Exp Biol Med 217: 188–196, 1998.
16. Eicher EM and Beamer WG. Inherited ateliotic dwarfism in
mice. Characteristics of the mutation, little, on chromosome 6.
J Hered 67: 87–91, 1976.
17. Faria AC, Veldhuis JD, Thorner MO, and Vance ML. Halftime of endogenous growth hormone (GH) disappearance in
normal man after stimulation of GH secretion by GH-releasing
hormone and suppression with somatostatin. J Clin Endocrinol
Metab 68: 535–541, 1989.
18. Fletcher TP, Thomas GB, and Clarke IJ. Growth hormonereleasing hormone and somatostatin concentrations in the hypophysial portal blood of conscious sheep during the infusion of
growth hormone-releasing peptide-6. Domest Anim Endocrinol
13: 251–258, 1996.
19. Frohman MA, Downs TR, Chomczynski P, and Frohman
LA. Cloning and characterization of mouse growth hormonereleasing hormone (GRH) complementary DNA: increased GRH
messenger RNA levels in the growth hormone-deficient lit/lit
mouse. Mol Endocrinol 3: 1529–1536, 1989.
20. Frohman LA, Downs TR, Clarke IJ, and Thomas GB. Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep.
Spontaneous secretion and response to insulin-induced hypoglycemia. J Clin Invest 86: 17–24, 1990.
21. Gaylinn BD, DeAlmeida VI, Lyons CE Jr, Wu KC, Mayo
KE, and Thorner MO. The mutant growth hormone-releasing
hormone (GHRH) receptor of the little mouse does not bind
GHRH. Endocrinology 140: 5066–5074, 1999.
22. Ghigo E, Arvat E, Gianotti L, Imbimbo BP, Lenaerts V,
Deghenghi R, and Camanni F. Growth hormone-releasing
activity of hexarelin, a new synthetic hexapeptide, after intravenous, subcutaneous, intranasal, and oral administration in
man. J Clin Endocrinol Metab 78: 693–698, 1994.
23. Giustina A and Veldhuis JD. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals
and the human. Endocr Rev 19: 717–797, 1998.
24. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins
NA, and Mayo KE. GHRH receptor of little mice contains a
missense mutation in the extracellular domain that disrupts
receptor function. Nat Genet 4: 227–232, 1993.
25. Gondo RG, Aguiar-Oliveira MH, Hayashida CY, Toledo
SP, Abelin N, Levine MA, Bowers CY, Souza AH, Pereira
RM, Santos NL, and Salvatori R. Growth hormone-releasing
peptide-2 stimulates GH secretion in GH-deficient patients with
mutated GH-releasing hormone receptor. J Clin Endocrinol
Metab 86: 3279–3283., 2001.
26. Gourlet P, Vandermeers A, Van Rampelbergh J, De Neef
P, Cnudde J, Waelbroeck M, and Robberecht P. Analogues
of VIP, helodermin, and PACAP discriminate between rat and
human VIP1 and VIP2 receptors. Ann NY Acad Sci 865: 247–
252, 1998.
27. Granda-Ayala R, Bowers CY, Parulkar A, Anand M, Baker
V, and Veldhuis JD. Continuous infusion of GHRP-2 for 30
days in elderly GH deficient subjects (Abstract). J Investig Med
48: 114A, 2000.
28. Hartman ML, Faria AC, Vance ML, Johnson ML, Thorner
MO, and Veldhuis JD. Temporal structure of in vivo growth
hormone secretory events in humans. Am J Physiol Endocrinol
Metab 260: E101–E110, 1991.
29. Hayashida CY, Gondo RG, Ferrari C, Toledo SP, Salvatori
R, Levine MA, Ezabella MC, Abelin N, Gianella-Neto D,
and Wajchenberg BL. Familial growth hormone deficiency
with mutated GHRH receptor gene: clinical and hormonal find-
282 • APRIL 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.247 on June 18, 2017
1. Aguiar-Oliveira MH, Gill MS, de Barretto AES, Alcantara
MR, Miraki-Moud F, Menezes CA, Souza AH, Martinelli
CE, Pereira FA, Salvatori R, Levine MA, Shalet SM, Camacho-Hübner C, and Clayton PE. Effect of severe growth
hormone (GH) deficiency due to a mutation in the GH-releasing
hormone receptor on insulin-like growth factors (IGFs), IGFbinding proteins, and ternary complex formation throughout life.
J Clin Endocrinol Metab 84: 4118–4126, 1999.
2. Arvat E, Gianotti L, Grottoli S, Imbimbo BP, Lenaerts V,
Deghenghi R, Camanni F, and Ghigo E. Arginine and growth
hormone-releasing hormone restore the blunted growth hormone-releasing activity of hexarelin in elderly subjects. J Clin
Endocrinol Metab 79: 1440–1443, 1994.
3. Barinaga M, Yamonoto G, Rivier C, Vale W, Evans R, and
Rosenfeld MG. Transcriptional regulation of growth hormone
gene expression by growth hormone-releasing factor. Nature
306: 84–85, 1983.
4. Baumann G and Maheshwari H. The Dwarfs of Sindh: severe
growth hormone (GH) deficiency caused by a mutation in the
GH-releasing hormone receptor gene. Acta Paediatr Suppl 423:
33–38, 1997.
5. Baumbach WR, Carrick TA, Pausch MH, Bingham B, Carmignac D, Robinson IC, Houghten R, Eppler CM, Price LA,
and Zysk JR. A linear hexapeptide somatostatin antagonist
blocks somatostatin activity in vitro and influences growth hormone release in rats. Mol Pharmacol 54: 864–873, 1998.
6. Billestrup N, Swanson LW, and Vale W. Growth hormonereleasing factor stimulates proliferation of somatotrophs in vitro.
Proc Natl Acad Sci USA 83: 6854–6857, 1986.
7. Calabresi E, Ishikawa E, Bartolini L, Delitala G, Fanciulli
G, Oliva O, Veldhuis JD, and Serio M. Somatostatin infusion
suppresses GH secretory burst frequency and mass in normal
men. Am J Physiol Endocrinol Metab 270: E975–E979, 1996.
8. Cataldi M, Magnan E, Guillaume V, Dutour A, ConteDevolx B, Lombardi G, and Oliver C. Relationship between
hypophyseal portal GHRH and somatostatin and peripheral GH
levels in the conscious sheep. J Endocrinol Invest 17: 717–722,
1994.
9. Chapman IM, Hartman ML, Straume M, Johnson ML,
Veldhuis JD, and Thorner MO. Enhanced sensitivity growth
hormone (GH) chemiluminescence assay reveals lower postglucose nadir GH concentrations in men than women. J Clin Endocrinol Metab 78: 1312–1319, 1994.
10. Chihara K, Kaji H, Minamitani N, Kodama H, Kita T, Goto
B, Chiba T, Coy DH, and Fujita T. Stimulation of growth
hormone by vasoactive intestinal polypeptide in acromegaly.
J Clin Endocrinol Metab 58: 81–86, 1984.
11. Cronin MJ, Hewlett EL, Evans WS, Thorner MO, and
Rogol AD. Human pancreatic tumor growth hormone (GH)releasing factor and cyclic adenosine 3⬘,5⬘-monophosphate evoke
GH release from anterior pituitary cells: the effects of pertussis
toxin, cholera toxin, forskolin, and cycloheximide. Endocrinology
114: 904–913, 1984.
12. Dimaraki EV, Jaffe CA, DeMott-Friberg R, Pan W, Brown
MB, and Barkan AL. Analysis of pulsatile hormone secretion:
relative importance of biological versus technical variability (Abstract). Prog Ann Mtg Endocrine Soc 82nd Toronto 2000, p. 487.
13. Dimaraki EV, Jaffe CA, Demott-Friberg R, Russell-Aulet
M, Bowers CY, Marbach P, and Barkan AL. Generation of
growth hormone pulsatility in women: evidence against somatostatin withdrawal as pulse initiator. Am J Physiol Endocrinol
Metab 280: E489–E495, 2001.
E949
E950
30.
31.
32.
33.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
ings in homozygous and heterozygous individuals from Itabaianinha. Eur J Endocrinol 142: 557–563, 2000.
Holl RW, Thorner MO, Mandell GL, Sullivan JA, Sinha YN,
and Leong DA. Spontaneous oscillations of intracellular calcium and growth hormone secretion. J Biol Chem 263: 9682–
9685, 1988.
Horikawa R, Fujita K, Nakajima R, Gaylinn B, and
Tanaka T. A novel growth hormone-releasing hormone (GHRH)
receptor mutation as a cause for isolated GH deficiency in a
Japanese boy with severe short stature (Abstract). Prog 82nd
Ann Mtg Endocrine Soc 82nd Toronto 2000, p. 482.
Huhn WC, Hartman ML, Pezzoli SS, and Thorner MO.
Twenty-four-hour growth hormone (GH)-releasing peptide
(GHRP) infusion enhances pulsatile GH secretion and specifically attenuates the response to a subsequent GHRP bolus.
J Clin Endocrinol Metab 76: 1202–1208, 1993.
Jaffe CA, DeMott-Friberg R, and Barkan AL. Endogenous
growth hormone (GH)-releasing hormone is required for GH
responses to pharmacological stimuli. J Clin Invest 97: 934–940,
1996.
Jaffe CA, Friberg RD, and Barkan AL. Suppression of
growth hormone (GH) secretion by a selective GH-releasing
hormone (GHRH) antagonist. Direct evidence for involvement of
endogenous GHRH in the generation of GH pulses. J Clin Invest
92: 695–701, 1993.
Jaffe CA, Ho PJ, Demott-Friberg R, Bowers CY, and Barkan AL. Effects of a prolonged growth hormone (GH)-releasing
peptide infusion on pulsatile GH secretion in normal men. J Clin
Endocrinol Metab 77: 1641–1647, 1993.
Jaffe CA, Turgeon DK, Friberg RD, Watkins PB, and Barkan AL. Nocturnal augmentation of growth hormone (GH) secretion is preserved during repetitive bolus administration of
GH-releasing hormone: potential involvement of endogenous somatostatin—a clinical research center study. J Clin Endocrinol
Metab 80: 3321–3326, 1995.
Kasting NW, Martin JB, and Arnold MA. Pulsatile somatostatin release from the median eminence of the unanesthetized
rat and its relationship to plasma growth hormone levels. Endocrinology 109: 1739–1745, 1981.
Kato Y, Shimatsu A, Matsushita N, Ohta H, and Imura H.
Role of vasoactive intestinal polypeptide (VIP) in regulating the
pituitary function in man. Peptides 5: 389–394, 1984.
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, and
Kangawa K. Ghrelin is a growth-hormone-releasing acylated
peptide from stomach. Nature 402: 656–660, 1999.
Laburthe M, Amiranoff B, Boige N, Rouyer-Fessard C,
Tatemoto K, and Moroder L. Interaction of GRF with VIP
receptors and stimulation of adenylate cyclase in rat and human
intestinal epithelial membranes. Comparison with PHI and secretin. FEBS Lett 159: 89–92, 1983.
Lin SC, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE, and
Rosenfeld MG. Molecular basis of the little mouse phenotype
and implications for cell type-specific growth. Nature 364: 208–
213, 1993.
Lindström P and Savendahl L. Effects of galanin on growth
hormone release in isolated cultured rat somatotrophs. Acta
Endocrinol 129: 268–272, 1993.
Lopez FJ, Meade EH Jr, and Negro-Vilar A. Development
and characterization of a specific and sensitive radioimmunoassay for rat galanin: measurement in brain tissue, hypophyseal
portal and peripheral serum. Brain Res Bull 24: 395–399, 1990.
Maheshwari HG and Baumann G. Genomic organization and
structure of the human growth hormone releasing hormone
receptor gene (Abstract). Prog Ann Mtg Endocrine Soc 79th
Minneapolis 1997, p. 156.
Maheshwari HG, Rahim A, Shalet SM, and Baumann G.
Selective lack of growth hormone (GH) response to the GHreleasing peptide hexarelin in patients with GH-releasing hormone receptor deficiency. J Clin Endocrinol Metab 84: 956–959,
1999.
Maheshwari HG, Silverman BL, Dupuis J, and Baumann
G. Phenotype and genetic analysis of a syndrome caused by an
inactivating mutation in the growth hormone-releasing hormone
AJP-Endocrinol Metab • VOL
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
receptor: dwarfism of Sindh. J Clin Endocrinol Metab 83: 4065–
4074, 1998.
Maiter DM, Hooi SC, Koenig JI, and Martin JB. Galanin is
a physiological regulator of spontaneous pulsatile secretion of
growth hormone in the male rat. Endocrinology 126: 1216–1222,
1990.
Martha PM, Blizzard RM, McDonald JA, Thorner MO, and
Rogol AD. A persistent pattern of varying pituitary responsivity
to exogenous growth hormone (GH)-releasing hormone in GHdeficient children: evidence supporting periodic somatostatin
secretion. J Clin Endocrinol Metab 67: 449–454, 1988.
Murray RA, Maheshwari HG, Russell EJ, and Baumann G.
Pituitary hypoplasia in patients with a mutation in the growth
hormone-releasing hormone receptor gene. ANJR Am J Neuroradiol 21: 685–689, 2000.
Netchine I, Talon P, Dastot F, Vitaux F, Goossens M, and
Amselem S. Extensive phenotypic analysis of a family with
growth hormone (GH) deficiency caused by a mutation in the
GH-releasing hormone receptor gene. J Clin Endocrinol Metab
83: 432–436, 1998.
Ocampo-Lim B, Guo W, DeMott-Friberg R, Barkan AL,
and Jaffe CA. Nocturnal growth hormone (GH) secretion is
eliminated by infusion of GH-releasing hormone antagonist.
J Clin Endocrinol Metab 81: 4396–4399, 1996.
Pincus SM. Approximate entropy as a measure of system complexity. Proc Natl Acad Sci USA 88: 2297–2301, 1991.
Pincus SM, Gevers EF, Robinson IC, van den Berg G,
Roelfsema F, Hartman ML, and Veldhuis JD. Females secrete growth hormone with more process irregularity than males
in both humans and rats. Am J Physiol Endocrinol Metab 270:
E107–E115, 1996.
Pincus SM and Keefe DL. Quantification of hormone pulsatility via an approximate entropy algorithm. Am J Physiol Endocrinol Metab 262: E741–E754, 1992.
Plotsky PM and Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal
circulation of the rat. Science 230: 461–463, 1985.
Rawlings SR and Hezareh M. Pituitary adenylate cyclaseactivating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary
gland. Endocr Rev 17: 4–29, 1996.
Rekasi Z, Czompoly T, Schally AV, and Halmos G. Isolation
and sequencing of cDNAs for splice variants of growth hormonereleasing hormone receptors from human cancers. Proc Natl
Acad Sci USA 97: 10561–10566, 2000.
Roelfsma F, Biermasz NR, Veldman RG, Veldhuis JD,
Frolich M, Stokvis-Brantsma WH, and Wit JM. Growth
hormone (GH) secretion in patients with an inactivating defect of
the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH
pulses. J Clin Endocrinol Metab 86: 2459–2464, 2001.
Salvatori R, Fan X, Phillips JA III, Espigares-Martin R,
Martin de Lara I, Freeman KL, Plotnick L, Al-Ashwal A,
and Levine MA. Three new mutations in the gene for the
growth hormone (GH)-releasing hormone receptor in familial
isolated GH deficiency type IB. J Clin Endocrinol Metab 86:
273–279, 2001.
Salvatori R, Hayashida CY, Aguiar-Oliveira MH, Phillips
JA III, Souza AH, Gondo RG, Toledo SP, Conceicao MM,
Prince M, Maheshwari HG, Baumann G, and Levine MA.
Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene. J Clin Endocrinol Metab
84: 917–923, 1999.
Sassolas G, Khalfallah Y, Chayvialle JA, Cohen R, Merabet S, Casez JP, Calvet P, and Cabrera P. Effects of the
somatostatin analog BIM 23014 on the secretion of growth hormone, thyrotropin, and digestive peptides in normal men. J Clin
Endocrinol Metab 68: 239–246, 1989.
Schriock EA, Hulse JA, Harris DA, Kaplan SL, and Grumbach MM. Evaluation of hypothalamic dysfunction in growth
hormone (GH)-deficient patients using single versus multiple
doses of GH-releasing hormone (GHRH-44) and evidence for
diurnal variation in somatotroph responsiveness to GHRH in
282 • APRIL 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.247 on June 18, 2017
34.
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
GH SECRETION IN GHRH RECEPTOR DEFICIENCY
63.
64.
65.
66.
68.
69.
70.
71.
72.
AJP-Endocrinol Metab • VOL
73.
74.
75.
76.
77.
78.
79.
80.
tion in normal man during a continuous 24-hour infusion of
human growth hormone releasing factor (1–40). Evidence for
intermittent somatostatin secretion. J Clin Invest 75: 1584–
1590, 1985.
Varga JL, Schally AV, Csernus VJ, Zarandi M, Halmos G,
Groot K, and Rekasi Z. Synthesis and biological evaluation of
antagonists of growth hormone-releasing hormone with high and
protracted in vivo activities. Proc Natl Acad Sci USA 96: 692–
697, 1999.
Veldhuis JD and Johnson ML. Cluster analysis: a simple,
versatile, and robust algorithm for endocrine pulse detection.
Am J Physiol Endocrinol Metab 250: E486–E493, 1986.
Veldhuis JD and Johnson ML. Deconvolution analysis of
hormone data. Methods Enzymol 210: 539–575, 1992.
Veldhuis JD and Johnson ML. Specific methodological approaches to selected contemporary issues in deconvolution analysis of pulsatile neuroendocrine data. Methods Neurosci 28:
25–92, 1995.
Waelbroeck M, Robberecht P, Coy DH, Camus JC, De Neef
P, and Christophe J. Interaction of growth hormone-releasing
factor (GRF) and 14 GRF analogs with vasoactive intestinal
peptide (VIP) receptors of rat pancreas. Discovery of (N-AcTyr1,D-Phe2)-GRF(1–29)-NH2 as a VIP antagonist. Endocrinology 116: 2643–2649, 1985.
Wajnrajch MP, Gertner JM, Harbison MD, Chua SC Jr,
and Leibel RL. Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat Genet 12: 88–90, 1996.
Webb CB, Vance ML, Thorner MO, Perisutti G, Thominet
J, Rivier J, Vale W, and Frohman LA. Plasma growth hormone responses to constant infusions of human pancreatic
growth hormone releasing factor. Intermittent secretion or response attenuation. J Clin Invest 74: 96–103, 1984.
Wehrenberg WB, Brazeau P, Luben R, Bohlen P, and
Guillemin R. Inhibition of the pulsatile secretion of growth
hormone by monoclonal antibodies to the hypothalamic growth
hormone releasing factor (GRF). Endocrinology 111: 2147–2148,
1982.
282 • APRIL 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.247 on June 18, 2017
67.
GH-deficient patients. J Clin Endocrinol Metab 65: 1177–1182,
1987.
Stewart JK, Clifton DK, Koerker DJ, Rogol AD, Jaffe T,
and Goodner CJ. Pulsatile release of growth hormone and
prolactin from the primate pituitary in vitro. Endocrinology 116:
1–5, 1985.
Stokvis-Brantsma WH, Blankenstein O, Oostdijk W,
Roelfsma F, Pfäffle R, and Wit JM. Endocrine findings and
growth response to combined GH and GnRH agonist treatment
in 2 siblings with a novel mutation of the GHRH receptor
(Abstract). Horm Res 53, Suppl 2: 57, 2000.
Tannenbaum GS and Ling N. The interrelationship of growth
hormone (GH)-releasing factor and somatostatin in generation of
the ultradian rhythm of GH secretion. Endocrinology 115: 1952–
1957, 1984.
Thomas GB, Cummins JT, Francis H, Sudbury AW, McCloud PI, and Clarke IJ. Effect of restricted feeding on the
relationship between hypophysial portal concentrations of
growth hormone (GH)-releasing factor and somatostatin, and
jugular concentrations of GH in ovariectomized ewes. Endocrinology 128: 1151–1158, 1991.
Thorner MO, Holl RW, and Leong DA. The somatotrope: an
endocrine cell with functional calcium transients. J Exp Biol
139: 169–179, 1988.
Treier M, Gleiberman AS, O’Connell SM, Szeto DP, McMahon JA, McMahon AP, and Rosenfeld MG. Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev
12: 1691–1704, 1998.
Van Cauter E, Caufriez A, Kerkhofs M, Van Onderbergen
A, Thorner MO, and Copinschi G. Sleep, awakenings, and
insulin-like growth factor-I modulate the growth hormone (GH)
secretory response to GH-releasing hormone. J Clin Endocrinol
Metab 74: 1451–1459, 1992.
Van Cauter E, Plat L, and Copinschi G. Interrelations between sleep and the somatotropic axis. Sleep 21: 553–566, 1998.
Van den Berg G, Frolich M, Veldhuis JD, and Roelfsma F.
Growth hormone secretion in recently operated acromegalic patients. J Clin Endocrinol Metab 79: 1706–1715, 1994.
Vance ML, Kaiser DL, Evans WS, Furlanetto R, Vale W,
Rivier J, and Thorner MO. Pulsatile growth hormone secre-
E951