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Departments of Internal Medicine, University of Michigan and Ann Arbor Veterans Affairs Medical Center, Ann Arbor, Michigan 48109; and the Department of Medicine, Tulane University (C.Y.B.), New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Craig A. Jaffe, M.D., Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Michigan Medical Center, 3920 Taubman, Ann Arbor, Michigan 48109-0354. E-mail: cjaffe{at}umich.edu
Abstract
GH-releasing peptide-6 (GHRP-6) is a potent GH secretagogue that releases GH by uncertain mechanisms. To assess whether GHRH is required for GH release by GHRP-6 in humans, we used the specific antagonist to GHRH (N-Ac-Tyr1,D-Arg2)GHRH(1–29)NH2 (GHRH Ant). We have previously shown that GHRH-Ant (400 µg/kg) blocked the GH response to 0.33 and 3.3 µg/kg boluses of GHRH by 95% and 81%, respectively. Nine healthy men between the ages of 20 and 30 yr were studied on two occasions. They received either saline or GHRH-Ant (400 µg/kg, iv) at 0840 h, followed by GHRP-6 (1 µg/kg, iv bolus) at 0900 h. Blood was sampled every 10 min from 0800–1100 h. GH responses were measured as the maximal increase over the baseline GH concentration and as the area under the curve. GHRH-Ant eliminated most of the GH response to GHRP-6 [maximal increase over the baseline GH concentration, 33.8 ± 4.8 vs. 6.2 ± 1.8 µg/L (mean ±SEM; P < 0.0001); area under the curve, 1701 ± 278 vs. 376 ± 113 µg/min·L (P < 0.001)]. These data show that endogenous GHRH is necessary for most of the GH response to GHRP-6 in humans.
THE SYNTHETIC hexapeptide, GH-releasing peptide-6 (GHRP-6; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), is an extremely potent stimulant of GH secretion in vivo and in vitro (1). GHRP-6 and other related peptides reliably stimulate GH secretion after iv, sc, intranasal, and even oral administration, thus rendering these compounds promising for clinical use as GH secretagogues (2, 3, 4).
The precise neuroendocrine mechanism(s) of action of the GHRPs is unclear. GHRP and GHRH act synergistically (2, 5) and independently of each other at the pituitary level via separate receptors (1, 6, 7) and intracellular messenger pathways (5, 8, 9). A specific G protein-coupled receptor for GH secretagogues has recently been cloned (7). In addition to direct pituitary action by the peptide, GHRPs require an intact hypothalamus for their full GH-releasing effect (6, 10). The nature of the hypothalamic component of the GHRP effect is unclear. We have previously shown that administration of a continuous infusion of GHRH to normal men abolished the acute GH response to GHRH, but preserved the GH response to GHRP (11). This suggested that the GH-releasing effect of GHRP was not mediated exclusively through GHRH release. On the other hand, animal data indicate that GHRH is crucial for the full effect of GHRP. In rats, for example, GHRP-6-induced GH release is reduced after prior administration of GHRH antiserum (10), and it has also been shown that dwarf mice with point mutations in the N-terminal ligand-binding domain of the GHRH receptor do not respond to either GHRH or GHRP (12).
The reduced efficacy of GHRP in stimulating GH release in children with isolated GH (presumably GHRH) deficiency (3), is the only circumstantial evidence in humans indicating the need for hypothalamic GHRH for the GHRP effect. The blunted GH response to GHRP in these patients could, however, be a manifestation of low or absent pituitary GH stores brought about by chronic GHRH deficiency, rather than a requirement for acute GHRH involvement. To differentiate these two possibilities, a model of acute GHRH deprivation is required, and until recently, no scientific tools were available to achieve this goal in humans.
In the present study, we examined the role of GHRH in the GH response to GHRP using a specific peptidic antagonist of GHRH (N-Ac-Tyr1,D-Arg2)GHRH(1–29)NH2 (GHRH-Ant). We have previously shown that GHRH-Ant suppresses the GH response to GHRH, clonidine, L-dopa, arginine, pyridostigmine, and hypoglycemia, but not to somatostatin withdrawal (13). Thus, GHRH-Ant is ideally suited to assess the involvement of endogenous GHRH in the generation of acute secretory GH responses to GHRP-6.
Materials and Methods
This study was approved by the University of Michigan institutional review board and the General Clinical Research Center review committee. An informed consent document was signed by all subjects before participation in the study. Nine men, aged 20–30 yr, of normal height (1.71–1.92 m) and body mass index (22.5–26.8 kg/m2) were recruited through newspaper advertisements. All were nonsmokers without any significant medical conditions and taking no medications. Their physical examinations were unremarkable, as were their complete blood counts and screening biochemical indexes.
Each subject acted as his own control and was studied on 2 separate days, receiving normal saline or GHRH-Ant in random order. The interval between the two studies was at least 7 days. The subjects were admitted to the General Clinical Research Center at 0730 h after an overnight fast, at which time an indwelling, heparin-filled, iv catheter was placed in a forearm vein. Blood sampling for measurement of plasma GH began at 0800 h and continued every 10 min until completion of the protocol 3 h later at 1100 h. The subjects remained fasting during this time. Normal saline (20 mL, iv) or GHRH-Ant (400 µg/kg BW, iv, over 6 min) was given at 0840 h on the control and treatment days, respectively. On both occasions, GHRP-6 (1 µg/kg BW) was administered as an iv bolus at 0900 h.
GHRP was synthesized by Peninsula Laboratories (Belmont, CA) under the Good Manufacturing Practice requirements of the FDA and was formulated at Tulane University (New Orleans, LA). GHRH antagonist was purchased from Bachem (King of Prussia, PA) and dissolved in normal saline to a final concentration of 5.0 mg/mL.
Plasma GH was measured in duplicate using immunochemiluminometric assay kits (Nichols Institute Diagnostics, San Juan Capistrano, CA). All samples from a given patient were run in the same assay. Assay sensitivity, using 50 µL plasma/tube was 0.01 µg/L, and the intraassay coefficient of variation was 6% at GH concentrations above 1 µg/L, below 6% at GH concentrations between 0.1–1.0 µg/L, and approximately 12% at GH concentrations below 0.1 µg/L.
The GH response to GHRP (
GH) was calculated as the difference
between the maximum GH concentration after GHRP administration and the
GH concentration at 0900 h (just before the GHRP bolus). The 2-h
integrated GH concentration was calculated as the area under the GH
vs. time curve (AUC), for the time period 0900–1100 h.
Paired t tests were used to compare the GH responses during
the 2 study days. Data are expressed as the mean ±
SEM. P < 0.05 was considered statistically
significant.
Results
After saline pretreatment, GHRP administration led to a marked
increase in plasma GH in all subjects, with peak GH concentrations
usually occurring within 40 min and returning to baseline within 80 min
(Fig. 1
). Prior administration of GHRH
antagonist blocked most of the GH secretion in response to GHRP (GH,
33.8 ± 4.8 vs. 6.2 ± 1.8 µg/L, control
vs. treatment; P < 0.0001). Similarly, the
2 h AUC decreased from 1701 ± 278 to 376 ± 113
µg/min·L (P < 0.001); Fig. 2 shows the mean GH response to GHRP on
control and treatment days. The average percent suppression was 77
± 5%. There was no correlation between the magnitude of the GH
response to GHRP-6 on the control day as measured by the 2 h AUC
and percent suppression by GHRH-Ant. Both GHRH-Ant and GHRP were well
tolerated. One subject felt slightly dizzy for 4 min after
administration of GHRP. Two subjects felt lightheaded, and one had a
warm flush after GHRH-Ant treatment. Each of these symptoms lasted less
than 2 min.
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This study shows that endogenous hypothalamic GHRH is necessary
for the acute GH-releasing effect of GHRP-6 to be fully manifest.
Nearly 80% of the GHRP-mediated GH release was suppressed by prior
administration of a specific GHRH antagonist. In six of the nine
subjects, virtually all GH secretion was eliminated by the antagonist,
whereas in the remaining three subjects, GH and the 2 h AUC
were measurable, although considerably attenuated. This incomplete
suppression raises the possibility that the dose of GHRH-Ant (400
µg/kg) was not adequate for all subjects. Although this is possible,
our previous data demonstrate that an identical dose of GHRH-Ant
eliminated 95% and 81% of the GH responses to physiological (0.33
µg/kg) and supramaximal (3.3 µg/kg) doses of GHRH, respectively
(13). Alternatively, the residual GH secretion in this study could have
reflected the purely pituitary, GHRH-independent mechanism of GHRP
action.
GHRPs probably release GH through both direct pituitary and hypothalamic effects. They clearly have a direct pituitary effect, as demonstrated by GHRP binding at the level of the pituitary (14, 15, 16), and GHRH-independent release of GH from somatotrophs in vitro (1, 17). However, multiple experimental paradigms support an additional hypothalamic site of binding and action (15, 16), which could account for their greater in vivo potency. Coincubation of GHRP and GHRH resulted in additive or synergistic GH release (5), suggesting that GHRH could play a role in GHRP-mediated GH release. This possibility was suggested by data from Dickson et al. (18) demonstrating that both peripheral as well as intracerebroventricular administration of GHRP activated a subpopulation of neurons in the arcuate nucleus of the hypothalamus (a known location of most GHRH-containing neuronal bodies), as reflected by increased electrical activity and an increase in Fos-like immunoreactivity in this area. Conceivably, activation of the arcuate nucleus by GHRP might result in the release of GHRH. This idea receives further support from the present study, from data demonstrating elimination of the GHRP effect in rodents after treatment with GHRH antiserum (10), and from direct measurements of the pituitary-portal GHRH concentration in sheep after systemic administration of another GHRP analog, hexarelin (19).
In an earlier study, we indirectly addressed the question of potential involvement of endogenous GHRH in the GH-releasing action of GHRP (11). Short term infusion of GHRH resulted in complete pituitary desensitization to a maximally effective dose of GHRH (1 µg/kg), whereas the GH rise to a bolus dose of GHRP was fully preserved. That study demonstrated that the GH-releasing effect of GHRP was not due exclusively to the putative GHRP-induced GHRH release. Our current data, on the other hand, show that endogenous hypothalamic GHRH is essential for maximal GH stimulation by GHRP. Thus, the two seemingly similar models of acute transient GHRH deprivation (homologous desensitization by a continuous agonist infusion and receptor blockade by a specific antagonist) reflect different facets of a complex neuroendocrine system.
These data are strikingly similar to the previously described effects of insulin hypoglycemia. Hypoglycemia-induced GH release is not attenuated and may even be potentiated by coadministration of a continuous GHRH infusion (20, 21). The GH response to hypoglycemia however, is severely attenuated by pretreatment with GHRH-Ant (13). It is possible that hypoglycemia (and potentially other hypothalamus-directed physiological and pharmacological GH secretagogues) causes acute release of an endogenous GHRP-like ligand as a significant component of its action. Conceivably, a cross-talk between the GHRH- and GHRP-activated intracellular cascades could account for the observed synergy and for the apparently conflicting results obtained by the paradigms of desensitization and receptor blockade.
GHRP has also been postulated to act as a functional somatostatin (SRIH) antagonist, based on its ability to abolish the refractoriness of the pituitary to repetitive GHRH boluses (10). The cellular mechanism for the anti-SRIH effect of GHRP is thought to be depolarization of Ca2+ and voltage-dependent K+ channels, which are normally kept in a hyperpolarized state by SRIH (22, 23). We have previously shown that GHRH-Ant does not attenuate the acute rebound GH rise that follows termination of SRIH infusion (13). Thus, had GHRP been acting solely or primarily as a SRIH antagonist, in effect creating a model of acute SRIH withdrawal, its effect would not have been expected to be blocked by GHRH-Ant. Functional SRIH antagonism, however, might account for much of the preserved GH response during GHRH-Ant treatment. The in vivo relationships between GHRP and SRIH have not been investigated in humans and require detailed studies in the future.
GHRPs have been shown to have only limited acute efficacy in individuals with GHRH deficiency (3). One obvious reason for this is the diminished GH stores in these subjects. Our study suggests yet another mechanism for this phenomenon: the dependence of the GHRP effect on the pituitary’s exposure to GHRH. The pituitary must be exposed to GHRH at the time of GHRP delivery in order for GHRP to be effective. Thus, we predict that in future long term studies, individuals with severely impaired GHRH secretion (e.g. structural hypothalamic damage, congenital panhypopituitarism, postirradiation, etc.) will increase their GH output to GHRP-like secretagogues only minimally or not at all. Newly developed GHRP-like preparations intended for clinical use need to be tested for their GHRH dependence using experimental paradigms similar to that described in this report.
In summary, our study demonstrates that GHRP in humans requires endogenous hypothalamic GHRH for its full GH-releasing effect to be manifest. The cellular mechanisms underlying this dependency are unknown.
Footnotes
1 This work was supported by Grant DK-38449 (to A.L.B.), a V.A. Merit
Review Award (to A.L.B.), Clinical Associate Physician Award
MO1-RR0043–34S3 (to C.A.J.), University of Michigan Diabetes Research
Training Center Award P60-DK20572 (to C.A.J.), and General Clinical
Research Center Grant MO1-RR-0042. 
Received November 6, 1997.
Accepted December 9, 1997.
References
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) and GHRH-Ant (•; 400
µg/kg) treatments.