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.


    Introduction
 Top
 Introduction
 References
 
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. Bartholomew’s 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 Turner’s 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, 0.41–2.2 U/mL).

Based on these results, the diagnosis of acromegaly was clear. It is of interest that the patient’s height was 5'3", which is very tall for a patient with Turner’s 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 1Go).


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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 )

 
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. Michael’s 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. 1Go); 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. 2Go). 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. 70:965–977, 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.

 
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 patient’s 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 {alpha} 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. 3Go) (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. 70:965–977, 1982.

 
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. Nature’s 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. Guillemin’s 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 group’s 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. 4Go). 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. 59:580–586, 1984.

 
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. 5Go (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. 24:145–151, 1988.

 
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. 6Go). 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.



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Figure 6. Individual height velocity (HV) for evaluable patients who completed 12 months of GHRH (1–29) treatment (30 µg/kg GHRH administered subcutaneously once a day at bedtime; n = 56). The HVs at baseline ({triangleup}) and after 6 months (•) and 12 months ({diamond}) of treatment are plotted by increasing baseline values. Note that there were better growth responses to GHRH in patients who had lower HVs at baseline, with the greatest response in the first 6 months of treatment. Reprinted with permission from Thorner et al., J Clin Endocrinol Metab. 81:1189–1196, 1996.

 
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. 7Go (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. 52:215–246, 1997.

 
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.


    Acknowledgments
 
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.


    Footnotes
 
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. Back

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.


    References
 Top
 Introduction
 References
 

  1. Reichlin S. 1961 Growth hormone content of pituitaries from rats with hypothalamic lesions. Endocrinology69 :225–230.
  2. Frohman LA, Szabo M, Berelowitz M, Stachura ME. 1980 Partial purification and characterisation of a peptide with growth hormone-releasing activity from extrapituitary tumors in patients with acromegaly. J Clin Invest. 65:43–54.[Medline]
  3. Thorner MO, Perryman RL, Cronin MJ, et al. 1982 Successful treatment of acromegaly by removal of a pancreatic islet tumor secreting a growth hormone-releasing factor. J Clin Invest. 70:965–977.[Medline]
  4. Cronin MJ, Rogol AD, Dabney LG, Thorner MO. 1982 Selective growth hormone and cyclic AMP stimulating activity is present in a human pancreatic islet cell tumor. J Clin Endocrinol Metab. 55:381–383.[Medline]
  5. Esch FS, Bohlen P, Ling NC, et al. 1982 Characterization of a 40 residue peptide from a human pancreatic tumor with growth hormone-releasing activity. Biochem Biophys Res Commun. 109:152–158.[Medline]
  6. Rivier J, Spiess J, Thorner M, Vale W. 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature. 300:276–278.[Medline]
  7. Spiess J, Rivier J, Thorner M, Vale W. 1982 Sequence analysis of a growth hormone-releasing factor from a human pancreatic islet tumor. Biochemistry. 24:6037–6040.
  8. Cronin MJ, Rogol AD, MacLeod RM, et al. 1983 Biological activity of a growth hormone-releasing factor secreted by a human tumor. Am J Physiol. 244:E346–E353.
  9. Thorner MO, Vance ML, Evans WS, et al. 1986 Physiological and clinical studies of GRF and GH. Recent Prog Horm Res. 42:589–640.[Medline]
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  14. Thorner MO, Rochiccioli P, Colle M, et al. 1996 Once daily subcutaneous growth hormone-releasing hormone therapy accelerates growth in growth hormone-deficient children during the first year of therapy. J Clin Endocrinol Metab. 81:1189–1196.[Abstract]
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  17. Mayo KE. 1992 Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. Mol Endocrinol. 6:1734–1744.[Abstract]
  18. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA, Mayo KE. 1993 GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat Genet. 4:227–232.[Medline]
  19. Lin SC, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE, Rosenfeld MG. 1993 Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature364 :208–213.
  20. Wajnrajch MP, Gertner JM, Harbison MD, Chua SC, Jr, Leibel RL. 1996 Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat Genet. 12:88–90.[Medline]
  21. Maheshwari HG, Silverman BL, Dupuis J, Baumann G. 1998 Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: Dwarfism of Sindh. J Clin Endocrinol Metab. 83:4065–4074.[Abstract/Free Full Text]
  22. Salvatori R, Hayashida CY, Aguiar-Oliveira MH, et al. 1999 Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene. J Clin Endocrinol Metab. 84:917–923.[Abstract/Free Full Text]
  23. Gaylinn BD, DeAlmeida VI, Lyons CE, Wu KC, Mayo KE, Thorner MO. 1999 The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH. Endocrinology. In press.