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Clinical Research Center Studies |
Department of Internal Medicine, Divisions of Endocrinology and Metabolism (C.A.J., R.D.-F., A.L.B), Department of Veterans Affairs Medical Center and University of Michigan Medical Center, Ann Arbor, Michigan 48109; and the Department of Internal Medicine, University of Illinois (L.A.F.), Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Craig A. Jaffe, M.D., Division of Endocrinology and Metabolism, 3920 Taubman Center, Box 0354, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0354.
Abstract
We have recently demonstrated that a competitive antagonist of GHRH, (N-Ac-Tyr1,D-Arg2)GHRH-(1–29)NH2 (GHRH-Ant), eliminates nearly all nocturnal GH pulsatility in normal subjects, supporting the hypothesis that GH pulsatility is driven by GHRH. In this study, we compared the effects of every 12 h iv boluses of either GHRH-Ant or saline on 24-h GH profiles in a patient with acromegaly due to a metastatic GHRH-secreting carcinoid tumor. Bolus doses of GHRH-Ant (400 µg/kg, iv) acutely suppressed GH concentration to 30–40% of the pretreatment baseline, and this effect lasted 3–4 h. Administration of GHRH (0.33 µg/kg, iv) bolus resulted in a small rise in GH, and this effect was blocked by GHRH-Ant (400 µg/kg). During saline treatment, the secretory patterns of both GH and ectopic GHRH were pulsatile; however, there was no correlation between changes in plasma GHRH and GH concentrations. This lack of correlation was probably due to the majority of circulating GHRH immunoreactivity consisting of nonbiologically active GHRH fragments. These data support the hypothesis that GH hypersecretion in the ectopic GHRH syndrome requires GHRH receptor occupancy and validates the use of GHRH-Ant to probe the potential involvement of endogenous GHRH in patients with acromegaly due to pituitary somatotropinoma.
THE ROLE OF hypothalamic GHRH in the regulation of GH secretion in humans is poorly defined. In rats (1) and sheep (2, 3, 4), GHRH secretion appears to be pulsatile and largely responsible for the generation of GH pulses. In humans, however, direct GHRH measurements in hypothalamic-pituitary portal plasma are impossible. Continuous infusion of GHRH results in an augmentation of GH pulsatility, suggesting that periodic SRIH decrements may be responsible for GH release (5). The potential involvement of endogenous GHRH in pituitary tumorogenesis and GH hypersecretion in patients with somatotropinomas has been suggested (6), although direct evidence in favor or against such a mechanism is lacking.
We have recently shown that a single injection of a specific antagonist to the GHRH receptor, (N-Ac-Tyr1,D-Arg2)GHRH-(1–29)NH2 (GHRH-Ant), eliminated 75% of the nocturnal GH pulsatility (7). We now report the use of GHRH-Ant to investigate the role of ectopic GHRH secretion in GH pulsatility in a patient with acromegaly due to a metastatic GHRH-secreting carcinoid tumor.
Materials and Methods
Subject and study design
The study was approved by the FDA, the University of Michigan institutional review board, and the General Clinical Research Center (GCRC) review committee, and an informed consent document was signed before the subject’s participation in the study. The subject was a 33-yr-old man with acromegaly due to ectopic GHRH production from a metastatic carcinoid tumor. Details of his clinical presentation and dynamic studies of GH and GHRH secretion have been previously reported (8). In brief, acromegaly was diagnosed in 1979, at which time he underwent a transsphenoidal partial hypophysectomy. In 1983, the diagnosis of ectopic GHRH syndrome was made. SRIH-14 suppressed both ectopic GHRH and GH, and since 1985 he had been treated with sc octreotide (1000 µg/day) and bromocriptine (20 mg/day), which resulted in normalization of both GH and insulin-like growth factor I, a decline in circulating GHRH levels, a decrease in pituitary size, and shrinkage of liver metastases. Octreotide and bromocriptine were discontinued 4 and 11 days, respectively, before the study.
The patient was admitted to the GCRC on the afternoon of day 1, at which time an iv catheter was placed in a forearm vein of one arm for blood sampling, and a second catheter was placed in a forearm vein of the other arm for drug administrations. Meals were served at 0700, 1300, and 1800 h, and there was no between-meal snacking. Water was allowed ad libitum. Throughout the study, the patient was allowed to walk freely in the GCRC, but he refrained from vigorous activity. Lights were turned on at 0700 h and off at 2300 h. Daytime napping was not permitted.
Spontaneous and GHRH-stimulated GH secretion were assessed during treatment first with saline and then with GHRH-Ant in an open label fashion. At 2000 h on the day of admission (day 1), 10 mL of the vehicle, normal saline, were given iv, and this was followed by the administration of GHRH (0.33 µg/kg BW) at 2100 h. Blood sampling for GH determination on day 1 was performed every 10 min from 1900–2400 h. At 0800 and 2000 on day 2, iv boluses of the vehicle were again given, and blood was sampled every 10 min for 24 h (0800–0800 h) for GH and GHRH measurements. Intravenous injections of GHRH-Ant (400 µg/kg BW) were given at 0800 and 2000 h on days 3 and 4. GHRH (0.33 µg/kg BW, iv bolus) was given at 2100 h, 1 h after GHRH-Ant administration. Blood was sampled for GH measurements on day 3, every 10 min from 0800–2400 h, and then for a 25-h time period (0700–0800 h) on days 4 and 5.
Assays
All plasma samples were diluted 1:10, and GH was measured in the same RIA, as previously described (7), with an assay detection limit of 0.3 µg/L and a mean coefficient of variation of 7%. Blood samples for GHRH measurement were collected into prechilled heparinized tubes containing aprotinin (1000 kallikrein inhibitor units/mL blood) and immediately centrifuged, and plasma was stored at -70 C until assayed. GHRH was extracted from the plasma samples and then measured by RIA as previously described (9). The cross-reactivity for GHRH-Ant in the GHRH assay was 100% on a molar basis.
Materials
GHRH-44 was purchased from Bachem (Torrance, CA) and prepared by the University of Michigan Investigational Pharmacy at a concentration of 50 µg/mL. GHRH-Ant was purchased from Bachem Bioscience (Philadelphia, PA) and prepared at a concentration of 5 mg/mL in 0.9% saline.
Data analysis
GH concentration peaks were defined by Cluster (10) using the parameters previously described (7). Cross-correlation analysis (11) of GH and GHRH was performed on the data obtained during the 24 h (0800–0800 h) of saline treatment. Lags of -100 to +100 min in 10-min increments were used.
Results
After saline pretreatment on day 1, there was a small (17 µg/L)
increase in plasma GH in response to the 2100 h GHRH injection
(data not shown). In contrast, after the 2000 h GHRH-Ant dose on
day 3, the GH concentration began to fall, and there was no increase in
plasma GH after the GHRH bolus. On both days, GHRH immunoreactivity at
the time of the GHRH bolus was high and did not increase appreciably
after GHRH administration. Figure 1
shows the 25-h GH
and GHRH concentration profiles during saline treatment on day 2.
Cluster analysis found 17 GH and 20 GHRH concentration peaks,
respectively. However, visual inspection of the plotted concentration
data, alignment of peaks defined by Cluster, and cross-correlation
analysis all failed to find any relationship between acute changes in
systemic GHRH immunoreactivity and the timing of GH pulses.
|
. After the first GHRH-Ant bolus, GH abruptly fell to
approximately 50% of the pretreatment level and remained suppressed
for approximately 3 h. During this same period, GHRH
immunoreactivity rose from 7 µg/L to greater than 100 µg/L due to
cross-reactivity with GHRH-Ant, and then declined with a half-life of
approximately 30 min (Fig. 3).
|
|
shows the 25-h (0700–0800 h) GH
profile during GHRH-Ant treatment on day 4. GH secretion was suppressed
for 3–4 h after GHRH-Ant administration, similar to the data shown in
the upper panel. Excluding the acute effects of the GHRH-Ant
bolus, the pattern of GH secretion was similar on all 3 days and was
marked by the presence of a large GH peak beginning at approximately
1300 h on each day. As shown in Fig. 1, there was
only a slight increase in systemic GHRH in samples obtained immediately
preceding this pronounced GH peak. The mean GHRH concentrations during
the one hour preceding the 1300 h GH peak, from 1300–1400 h and
from 1400–1500 h, were not significantly different. Discussion
The results of studies based on sampling of hypophysial-portal blood (1, 2, 3, 4) and immunoneutralization of GHRH in animals (1, 12) suggest that GH pulses are the result of acute GHRH release. Similarly, the demonstration that GHRH-Ant eliminates GH pulsatility in humans is consistent with the hypothesis that GH pulses in humans are driven by GHRH (7). In contrast, other data, such as the persistence of GH pulsatility during the continuous infusion of GHRH (5), have been cited as support for GHRH being more tonically secreted and for GH pulses to be the result of acute decreases in hypothalamic SRIH secretion into hypophysial-portal blood. GHRH is required for the development of GH hypersecretion and acromegaly in patients with the ectopic GHRH syndrome, and a good correlation between GH and GHRH pulses was observed in an individual patient with this syndrome and a single GHRH-secreting tumor (13). Treatment with somatostatin suppresses GH secretion in these patients, probably through a combination of suppression of GHRH release from neuroendocrine cells and direct suppressive effects on the somatotrophs (8, 13, 14).
One of the aims of this study was to investigate the relationship between GH pulses and circulating GHRH. The secretion of both hormones was clearly pulsatile; however, on cross-correlation analysis, there was no interrelationship. There are several reasons why we might not have been able to establish a correlation. First, the involvement of other GH secretagogues or a periodic decline in SRIH secretion must be considered, although these mechanisms were not directly assessed. It is also conceivable that the complicated and uncoordinated GHRH secretory pattern from multiple tumor sites could have obscured the relationship between GH and GHRH. Changes in pituitary responsiveness to GHRH might have also played a role. The administration of GHRH in doses that result in circulating GHRH concentrations comparable to those reported in this patient can result in homologous desensitization in normal subjects (15, 16). Clearly, despite massively increased GHRH concentrations, GH hypersecretion continued in this patient. However, it is possible that there were periods of relative desensitization after GHRH release from one of the metastases. More likely, our failure to establish this relationship was at least in part the result of GHRH-(1–44) undergoing rapid N-terminal dipeptide cleavage to form a more stable, biologically inactive fragment (17). In another patient with ectopic GHRH, the vast majority of plasma GHRH immunoreactivity was demonstrated by quantitative high performance liquid chromatography to be GHRH-(3–40) (18), and in other patients with the syndrome GHRH-(3–44) was predominant (our unpublished data).
In our previous studies in normal men, we showed that GHRH-Ant blocked the GH response to exogenous GHRH (7, 19). In the present study, this effect was again demonstrated. During the control treatment, there was a modest increase in GH in response to exogenous GHRH. This response was considerably smaller than the GH response to 1 µg/kg GHRH previously reported in this patient (8). However, the GHRH dose used in the present study was lower than that in the previous report. In addition, GHRH during control treatment happened to be administered after a time of maximal GHRH and GH secretion, and there might have been some degree of acute homologous desensitization. The dose of GHRH used would be expected to increase plasma GHRH immunoreactivity by approximately 2 µg/L (15), whereas plasma GHRH immunoreactivity at the time of GHRH injection on day 1 was approximately 20 µg/L. Failure to see an obvious increase in GHRH immunoreactivity after the GHRH bolus was probably a result of the very high endogenous GHRH concentrations on day 1 and the cross-reactivity of GHRH-Ant on day 3 obscuring the contribution of exogenous GHRH.
On all 3 days of the study when frequent blood sampling was performed, there was a large GH peak at approximately 1300 h. These peaks could have been the response to a meal, although the fact that similar changes did not occur around the morning or evening meals makes this unlikely. It is interesting that there was no clear nocturnal augmentation of GH secretion. Whether the midday peak represented a phase shift of the normal augmentation is unknown. It has been previously reported that patients with acromegaly had reproducible 24-h GH secretory patterns when they were studied on several occasions (20, 21). The mechanisms resulting in a stable circadian rhythm in patients with pituitary adenoma and in our patient with ectopic GHRH secretion are unknown.
Administration of GHRH-Ant abolished the response to exogenous GHRH and reproducibly suppressed GH hypersecretion on every occasion. The latter effect lasted 3–4 h and decreased GH to a plateau of approximately 30–40% of the pretreatment concentration. The limited duration of the suppression probably reflects clearance of the antagonist and the grossly elevated ectopic GHRH concentrations. Indeed, a negative relationship between the dose of exogenous GHRH and the duration of GHRH-Ant action has been previously noted by us (7). Thus, it is possible that even larger doses of GHRH-Ant are needed to overcome the GH-releasing effects of the ectopic GHRH. An alternative explanation is that long term exposure to ectopic GHRH induced a certain degree of autonomy in hyperplastic somatotrophs, similar to the development of tertiary hyperparathyroidism in chronic renal failure, where prolonged parathyroid stimulation initially leads to hyperplasia, then to monoclonal adenoma formation and autonomous secretion of PTH (22).
The involvement of endogenous GHRH in the development of pituitary somatotropinomas and the maintenance of GH hypersecretion has been debated (23). In support of this hypothesis is the development of somatotroph adenomas in patients with hypothalamic GHRH-secreting gangliocytomas (24, 25) and in mice transgenic for GHRH (26). However, direct proof of a role for GHRH in the pathogenesis of acromegaly is lacking. Our study validates GHRH-Ant as a tool to probe the potential role of GHRH in patients with acromegaly due to GH-secreting adenomas.
Acknowledgments
We thank the staff of the General Clinical Research Center for their excellent nursing and technical support, Kevin McFarland for his technical support, and Dave Mauger, Ph.D., and Wensheng Guo, B.S., for their assistance with the statistical analyses.
Footnotes
1 This work was supported by Grants DK-08729 (to C.A.J.),
M01-RR-0043-34S3 (Clinical Associate Physician Award, to C.A.J.),
DK-38449 (to A.L.B.), M01-RR-0042 (to the General Clinical Research
Center), and DK-30667 (to L.A.F.). 
Received August 12, 1996.
Revised October 18, 1996.
Accepted October 28, 1996.
References
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  J. Iqbal, T. R. Manley, P. Ciofi, and I. J. Clarke Reduction in Adiposity Affects the Extent of Afferent Projections to Growth Hormone-Releasing Hormone and Somatostatin Neurons and the Degree of Colocalization of Neuropeptides in Growth Hormone-Releasing Hormone and Somatostatin Cells of the Ovine Hypothalamus Endocrinology, November 1, 2005; 146(11): 4776 - 4785. [Abstract] [Full Text] [PDF]
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 M. M. Skinner, R. Nass, B. Lopes, E. R. Laws, and M. O. Thorner Growth Hormone Secretagogue Receptor Expression in Human Pituitary Tumors J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4314 - 4320. [Abstract] [Full Text]
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