The Discovery of Growth Hormone-Releasing Hormone1
MICHAEL O. THORNER
Department of Medicine, University of Virginia, Charlottesville,
Virginia 22908
Address correspondence and requests for reprints to: Michael O. Thorner, Department of Medicine, University of Virginia Health System, Box 466, Charlottesville, Virginia 22908.
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Introduction
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DR. EDWIN B. ASTWOOD was a visionary in
modern endocrinology. The Biographical Memoir by Roy Greep
and Monte Greer ended "In addition to his specific research
contributions, Dr. Edwin B. Astwood succeeded to an extent achieved by
few in spanning the broad area between Natural Sciences and clinical
medicine." To me, this is the highest calling in endocrinology.
The late 1960s and early 1970s were marked by the incredible
achievements of two groups, those of Drs. Andrew Schally and Roger
Guillemin, who isolated, characterized, and sequenced the hypothalamic
regulatory hormones. The first hypothalamic regulatory hormone to be
sequenced was TRH (a tripeptide), the second was gonadotropin-releasing
hormone (a decapeptide), and the third was somatostatin with 14 amino
acids. The existence of the GHRH had been suggested in the early 1960s
by some classical experiments by Reichlin (1); he made lesions in the
hypothalamus and demonstrated that the content of GH in the pituitary
declined. Based on these data, he suggested that there must be a GHRH
in the hypothalamus.
I did my medical training in London, England, with my endocrine
training under Prof. G. Michael Besser. One of the most enjoyable
conferences we attended on a regular basis was the presentation of
clinical cases at the Royal Society of Medicine. Etched in my mind was
the fact that every time a patient with acromegaly was presented, Prof.
Peter Sonksen would ask whether or not the patient had any evidence of
a carcinoid tumor. The perception in the audience was "Why is this
man always asking this question?" because such cases probably do not
exist or, if so, very rarely!
In 1977, I moved from St. Bartholomews Hospital (London, England) to
the University of Virginia. My main interest at that time was
PRL-secreting pituitary tumors and their medical treatment.
In October 1980, a 21-yr-old patient was referred to me by Dr. Ann
Johanson. This patient had amenorrhea and galactorrhea while taking an
oral contraceptive for gonadal steroid replacement. She had gonadal
agenesis from Turners syndrome.
Her evaluation revealed an enlarged pituitary fossa on skull x-ray, a
pituitary mass with modest suprasellar extension on computed tomography
scan, and an elevated serum PRL of 68 µg/L. Her elevated serum GH of
95 µg/L did not suppress with oral glucose, increased with TRH, and
suppressed with a dopamine infusion. Her serum insulin-like growth
factor (IGF)-I level was elevated at 11 U/mL (normal, 0.412.2
U/mL).
Based on these results, the diagnosis of acromegaly was clear. It is of
interest that the patients height was 5'3", which is very tall
for a patient with Turners syndrome. In this regard, it was fortunate
that sera had been saved from the previous 7 yr. Her serum GH
concentrations had been elevated throughout that time period (Table 1
).
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Table 1. Basal serum GH levels for the 10-yr period before
the diagnosis of acromegaly was established in a patient with a
pancreatic tumor secreting GHRH (3 )
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The patient was referred for transsphenoidal surgery, which was
performed on January 15, 1981. Seven weeks postoperatively, she was
shown to still have acromegaly; serum GH was 52 µg/L, and IGF-I was
still elevated at 7.7 U/mL.
As part of an ongoing collaboration, the pituitary pathology specimens
had been sent to Dr. Kalman Kovacs (St. Michaels Hospital,
Toronto, Canada). Sometime later, Dr. Kovacs called to give me the
results of his examination of the tissue. His report indicated that
this patient had somatotroph hyperplasia and not a pituitary
tumor. The reasons leading to this diagnosis were: 1) the
reticulin pattern was preserved (it is destroyed in tumors) (Fig. 1
); 2) somatotrophs predominated,
although thyrotrophs, gonadotrophs, and corticotrophs were
interspersed; and 3) electron microscopy of the somatotrophs revealed
well-developed endoplasmic reticulum and Golgi apparatus, suggestive of
active hormone synthesis (Fig. 2
).
Because the somatotrophs were densely packed with secretory granules,
this suggested to me that this reflected the inhibition of hormone
release due to feedback of secreted GH, presumably by enhancing
somatostatin secretion (and/or IGF-I).

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Figure 1. Reticulin staining of pituitary tissue removed
from a patient with ectopic GHRH secretion showing the pituitary
divided by reticulin fibers into neat acini. The hematoxylin-eosin (A)
and Gordon-Sweet silver stain (B) conclusively reveal the preservation
of the acinar structure and the enlargement of acini. The
immunostaining (C) shows that the majority of cells are GH
immunoreactive. Panel B is reprinted with permission from Thorner
et al., J Clin Invest. 70:965977, 1982. Panels A and C are
compliments of Dr. K. Kovacs.
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Figure 2. Electron micrograph of the pituitary lesion from a
patient with ectopic GHRH secretion showing a somatotroph with
unusually large and active Golgi complex and numerous secretory
granules, reflecting active hormone synthesis and inhibition of hormone
release, presumably by somatostatin (and/or IGF-I). Magnification,
x6,950. Compliments of Dr. K. Kovacs.
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Based on pituitary histology, it was clear that the pituitary was being
stimulated by an extrinsic factor. It was unlikely that this was due to
a mutation in the GHRH receptor or a constitutive activation of the G
protein coupled to the GHRH receptor because this likely would occur in
a single cell, leading to tumor development and the breakdown of the
reticulin pattern. Therefore, it was much more likely that the
pituitary was being stimulated by an extrinsic factor, either GHRH
coming from the hypothalamus or from the periphery. Secondly, the
persistent acromegaly made it imperative that we find the cause of this
patients acromegaly. At this time an article by Frohman et
al. (2) reported the partial purification and characterization of
a peptide with GH-releasing activity from extrapituitary tumors
from acromegalic patients.
An abdominal computed tomography scan demonstrated a 5-cm diameter
tumor in the tail of the pancreas. On August 26, 1981, the tumor was
removed. It was well encapsulated and on histology was determined to be
an islet cell tumor. However, the tumor was neither a ß nor
cell
tumor. It was strongly positive for neuron-specific enolase, and on
immunostaining was negative for GH, insulin, glucagon, calcitonin, and
ACTH, with equivocal staining for somatostatin.
The tumor contained no GH by immunocytochemistry. In the first 2 h
following tumor removal there was a rapid fall in serum GH levels from
70 µg/L to 3 µg/L (Fig. 3
) (3). The
patient has now been followed for 18 yr and remains cured of her
acromegaly.

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Figure 3. Serum GH and PRL concentrations during and after
removal of pancreatic tumor secreting GHRH. Note the rapid fall of GH
levels with no fall of PRL levels following removal of the tumor.
Modified and reprinted with permission from Thorner et
al., J Clin Invest. 70:965977, 1982.
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The next question was what to do with the pancreatic tumor. At that
time I was fortunate to have the late Dr. Michael Cronin as a colleague
at the University of Virginia. Michael came to the operating room with
me to help implement our carefully crafted strategy for handling the
tumor. Half of the tumor was diced and immediately put into liquid
nitrogen, and the other half was prepared for tissue culture. The
latter was sent to several groups who were experts in establishing
stable cell lines. Michael Cronin was able to demonstrate that the
medium that bathed this tumor contained GHRH activity and stimulated
adenylate cyclase (4). The tumor was initially provided to Dr. Wylie
Vale at the Salk Institute (La Jolla, CA), and he subsequently
gave a portion of it to Dr. Guillemin. The two groups independently
found that it contained high GHRH activity and were able to isolate the
GHRH from the tumor, characterize it, and sequence it (5, 6, 7, 8). GHRH from
this tumor was a 40-amino acid peptide, which has homology with members
of the glucagon/secretin/VIP family of peptides.
I chose endocrinology as my specialty because my mentors impressed on
me the importance of the application of physiology to the understanding
of pathophysiology and therapy of patients with endocrine disease.
Each patient that one sees is an experiment of nature. I was very
fortunate to be referred this patient who enabled me and my colleagues
to provide the tumor to two leading groups who were then able to
isolate, characterize, and sequence the long-sought GHRH. In fact, GHRH
was one of the first hypothalamic hormones to be sought, but there had
been many false starts.
There were several reasons why it was possible in 1982 to successfully
characterize and sequence GHRH. First, the methods for assessing the
activity of tumor extracts had been refined and were much more
sensitive. Second, methods for microsequencing were greatly improved
over the previous 10 yr. But the third, and probably the most
important, reason was that this tumor contained high concentrations of
GHRH and very little somatostatin. Somatostatin inhibits GH release in
the bioassay, thus, its presence confounded the monitoring of the
purification of GHRH. The extract from this tumor greatly simplified
the process (because it had high concentrations of GHRH and little
somatostatin was present), compared to earlier attempts using hundreds
of thousands of animal hypothalami that were rich in somatostatin and
had very small concentrations of GHRH. This was the first example of a
single human tumor used to identify and isolate a hormone. PTH-related
protein was isolated with a similar technique using a human tumor cell
line.
As clinicians we all have the responsibility to identify experiments of
nature and seize the opportunities to ask questions that would be very
difficult to answer in any other way. Natures experiments are often
unique, and certainly the 15 or 20 patients who came before this one
provided clues that a tumor from a patient with ectopic GHRH secretion
could answer the question of the nature and structure of GHRH. In fact,
Dr. Guillemins group subsequently became aware of another tumor from
Dr. Genevieve Sassolas (Centre de Medecine Nucleaire,
Hôpital Cardiologique, Lyon, France), and as they had more tissue
from that tumor, they studied it more aggressively than the one from
Charlottesville. However, it was the original Charlottesville tumor
that suggested that such a tumor might offer the key to the quest for
GHRH.
The subsequent study of the physiology of GHRH and its therapeutic
potential as a treatment for GH deficiency in children has been the
focus of our groups work over the last 17 yr (9, 10). Initially, we
administered GHRH to normal volunteers and demonstrated its efficacy
and specificity in increasing serum GH concentrations (11). We then
determined the GH response to an acute injection of GHRH in children
with idiopathic GH deficiency (Fig. 4
).
Many of the children had a very robust increase in GH, whereas in some
there was a small or no increase (12); the response to GHRH was greater
than to arginine and L-dopa. Based on these results, we
proposed that many children with idiopathic GH deficiency really suffer
from GHRH deficiency.

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Figure 4. GH release in response to an iv injection of 3.3
µg/kg as a bolus dose of GHRH (hpGRF-40) in children with short
stature. The bars at the lower right are the mean ± SEM of
the peak GH responses to arginine/L-dopa and GHRH
stimulation tests. Reprinted with permission from Rogol et
al., J Clin Endocrinol Metab. 59:580586, 1984.
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A multicenter trial of GHRH treatment was then initiated. This seemed
particularly appropriate because the GH used to treat GH deficiency was
derived from cadavers and had been implicated in the transmission of
Jacob Creutzfeld disease. A total of 24 children were treated; in 10
children a pump was used to administer a sc GHRH bolus every 3 h.
This route and dose were chosen to mimic the normal physiology of
approximately one GH pulse every 3 h in growing children. In
addition, 10 children were treated with the pump every 3 h
overnight only, and 4 children received twice daily sc injections of
GHRH. The growth velocities in the children before and at 6 months on
treatment are shown in Fig. 5
(13).

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Figure 5. The effect of three different treatment regimens
of GHRH therapy in GH-deficient children on growth velocity
(cm/yr). Reprinted with permission from Thorner et al.,
Pediatr Res. 24:145151, 1988.
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Based on the results of this study, Serono Laboratories, Inc.
initiated a 12-month multicenter study with GHRH(1-29)-NH2
administered sc once daily (14). The children who were growing most
slowly had the greatest increase in growth velocity (Fig. 6
). This suggests that GHRH may restore
physiologic GH secretion. GHRH is approved by the Food and Drug
Administration for diagnostic use, as well as for treatment of GH
deficiency in children. GHRH is safe and effectively accelerates growth
velocity in the majority of GH-deficient patients.
At the time GHRH was synthesized there was a great deal of interest in
the down-regulation of the gonadotropin response to the
gonadotropin-releasing hormone. We were intrigued by an observation in
another patient with ectopic GHRH secretion from a metastatic carcinoid
tumor that gave rise to acromegaly. Despite persistently elevated GHRH
levels throughout the 24-h period, the pattern of GH release was still
pulsatile, indicating that there are presumably other factors that are
involved in regulating the pulsatile pattern of GH secretion (15). It
has been suggested that this may be due to the withdrawal of
somatostatin. Thus, ectopic GHRH secretion unequivocally demonstrates
that a long-acting GHRH agonist can increase pulsatile GH secretion.
Continuous iv GHRH infusion studies in normal subjects demonstrate the
same phenomenon, and, thus, it is clear that a depot preparation of
GHRH may be a useful therapeutic agent.
Our group (16) and Mayo (17) cloned the GHRH receptor. It is
interesting that the GHRH receptor is a member of the B class of G
protein-coupled receptors, which includes the receptors for the
glucagon/secretin family of peptides, as well as CRH, calcitonin, and
parathyroid hormone. Their structure is totally different from that of
the larger rhodopsin (A family) of receptors. However, there are also
seven transmembrane-spanning domains in this family of receptors.
The critical role of GHRH is demonstrated by the dwarf lit
mouse that has a point mutation in the GHRH receptor (18, 19).
Wajnrajch et al. (20) described a nonsense mutation (Glu
72stop) in the human GHRH receptor causing growth failure and GH
deficiency in two children from the same Indian Moslem kindred.
Maheshwari et al. (21) have described a kindred in the
Valley of Sindh in Pakistan with the same mutation. Members of this
kindred are of extreme short stature. Another mutation in the receptor
has been described in a large Brazilian kindred that includes over 110
individuals affected with severe GH deficiency and extreme short
stature (22). These experiments of nature demonstrate that a defect in
the GHRH receptor impairs normal growth and emphasizes the vital
importance of GHRH.
A cartoon of the GHRH receptor is shown in Fig. 7
(10). Aspartic acid at position 60 from
the start codon is the site of the lit mouse mutation.
Western blotting using a C-terminal GHRH receptor antiserum that was
raised to a peptide based on the sequence of the human GHRH receptor
was used to study membranes from HEK293 cells transfected with
complementary DNA for the GHRH receptor. The studies indicate
that the lit mouse receptor is translated into protein and
is actually present in the membrane of pituitary cells in the
lit mouse. We have proposed that this mutation leads to a
change in the tertiary structure of the receptor, which prevents
binding (23). Using photo affinity probes, our evidence suggests that
other sites are in close proximity to bound GHRH, indicating that
several sites of interaction are probably required for signal
transduction. The availability of recombinant receptor makes the search
for nonpeptide GHRH agonists feasible.

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Figure 7. Cartoon of a hypothetical model of the GHRH
receptor illustrating the extracellular domains, transmembrane domains,
and the intracellular domains. The D represents ASP60, which is the
site of the point mutation in the lit mouse. Reprinted with
permission from Thorner et al., Recent Prog Horm Res.
52:215246, 1997.
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In conclusion, knowledge gleaned from careful observation of a single
patient with acromegaly led to the isolation, characterization, and
synthesis of GHRH. Additional studies led to the introduction of GHRH
treatment of GH deficiency. Subsequently, we and others were able to
clone the receptor for GHRH. Studies with GHRH have confirmed that it
is essential for GH synthesis and normal growth. This position is now
validated by observations that mutations of the GHRH receptor, which
result in its inactivation, lead to profound GH deficiency and severe
short stature and complete the circle of our understanding of GH
regulation by GHRH.
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Acknowledgments
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I thank my many colleagues who have made important contributions
to this work, as well as Ms. Suzan Pezzoli for assistance in
preparation of this manuscript.
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Footnotes
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1 This article is dedicated to the memory of Michael J. Cronin, Ph.D.,
my colleague, friend, and collaborator, who made such important
contributions to this story. 
This work was supported in part by NIH Grants HD-13197, DK-32632, and
DK-45350 (to M.O.T.) and Grant RR-00847 (to the General Clinical
Research Center and CDMAS Laboratory at the University of
Virginia.
Received September 13, 1999.
Accepted September 20, 1999.
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