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Clinical Research Center Studies |
Department of Medicine, Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center (S.E.K., M.L.H., S.S.P., M.O.T.), Charlottesville, Virginia 22908; University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School (S.R.S., J.R.S.), New Brunswick, New Jersey 08903; and Merck Research Laboratories (B.J.G., J.M.W., D.A.K.), Rahway, New Jersey 07065
Address all correspondence and requests for reprints to: Dr. Michael O. Thorner, Department of Medicine, Box 511–66, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail MOT{at}virginia.edu
Abstract
GH secretion and the response to GH secretagogues are significantly
diminished in obese individuals. Previous studies have shown that
L-692,429 (L), a nonpeptide mimetic of GH-releasing peptide,
selectively stimulates GH release in normal young men and in the
elderly, who also have diminished GH secretion. A paired, two-site
study examined the effects of L on GH release in 12 healthy obese (part
A; mean ± SD: age, 26.1 ± 3.3 yr; body mass
index, 35.0 ± 3.1 kg/m2) and 10 nonobese (part B;
age, 22.2 ± 2.3 yr; body mass index, 
27.0) young men. In part
A, placebo, low dose L (0.2 mg/kg), or high dose L (0.75 mg/kg) was
administered iv over 15 min on 3 separate occasions after an overnight
fast. Samples for GH, PRL, and cortisol determinations were obtained
every 15 min. GH release (mean ± SE) was
significantly increased by both doses of L compared to the effect of
placebo: 12.6 ± 1.8 µg/L (low dose), 18.5 ± 2.7 µg/L
(high dose), and 0.84 ± 0.1 µg/L (placebo), respectively
(P < 0.05). In a subset of 6 obese men, in samples
collected every 5 min, the GH response to both doses of L was
significantly greater than that to 1 µg/kg GHRH. To compare the
response to low dose L in the obese and to determine the effects of
feeding on this response, 0.2 mg/kg L was administered as described in
part A to nonobese young men after an overnight fast (fasted) or a
standardized breakfast (fed; part B). Low dose L was an effective GH
secretagogue in nonobese young men; however, this effect was attenuated
with feeding [43.6 ± 7.9 (fasted) vs. 17.7
± 4.8 (fed)µg/L]. Of note, the response to low dose L in fasted
obese individuals was similar to that in fed nonobese individuals. The
administration of L was well tolerated in both groups. We conclude that
L is an effective GH secretagogue in obese and nonobese young men and
may have therapeutic benefits when administered to relative (obese or
elderly) or absolute GH-deficient individuals.
THE REGULATION of GH secretion occurs by input from the hypothalamic factors GHRH and somatostatin. GHRH stimulates the transcription of GH messenger ribonucleic acid and hormone release, whereas somatostatin suppresses GH release but has no effect on gene expression (1). GH is also regulated by feedback at the level of the pituitary by systemic insulin-like growth factor I (IGF-I) (2, 3). The enhanced GH secretion observed during fasting in humans is quickly abolished with meals or iv administration of IGF-I (3, 4). Moreover, in obese individuals, spontaneous GH secretion is attenuated (5), and the GH response to secretagogues (GHRH, insulin-induced hypoglycemia, and arginine) is markedly diminished (6, 7), a defect that is corrected by weight loss and fasting (5, 8). It is unclear whether the alterations in GH secretion that occur in obese individuals are physiological or pathological.
GH-releasing peptide (GHRP) is a synthetic hexapeptide analog of met-enkephalin that selectively stimulates GH release by acting at both the hypothalamus and pituitary (9). Studies have shown that GHRP acts via a GH secretagogue receptor that is unrelated to enkephalin, somatostatin, and GHRH receptors (10, 11); additionally, it appears to act as a functional somatostatin antagonist via postreceptor mechanisms (12). The GH-releasing actions of GHRP are synergistic with GHRH (13), but in vivo studies in sheep have also shown that it increases GHRH release (14). Importantly, GHRP is effective with oral administration, although its bioavailability via this route is limited (15). The search for nonpeptide mimics of GHRP led to the synthesis of L-692,429 (L), which has GH-secretagogue activity indistinguishable from that of GHRP-6 both in vivo and in vitro (16). In initial studies, it was a well tolerated, selective GH secretagogue in young lean men (17). Moreover, in a group of healthy elderly subjects, a population in which GH secretion is diminished (18), L was a more potent GH secretagogue than GHRH (19). Finally, when administered simultaneously with prednisolone in healthy young men, L reversed the GH suppression normally seen with glucocorticoid administration (20).
The results from our earlier investigation with L in the elderly showed promise for the use of nonpeptidyl mimics of GHRP as therapeutic agents in a population with reduced GH secretion (19). Therefore, we designed the following study to determine the effects of L in two clinical situations in which GH secretion is attenuated: obese individuals and nonobese individuals in the postprandial state.
Subjects and Methods
Subjects
This paired study was carried out at two sites, the University of Virginia (part A: obese individuals) and the University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (UMDNJ; part B: nonobese individuals). The study protocols were approved by the University of Virginia General Clinical Research Center (GCRC) and the human investigation committee (part A); the institutional review board of UMDNJ (part B); and Merck Research Laboratories (parts A and B). Subjects were recruited by means of local advertisement and gave written informed consent before enrollment.
In part A, 12 obese men [body mass index (BMI), 
30] were studied
with a mean age of 26.1 yr (SD = 3.3; range, 20–35), a
mean BMI of 35.0 kg/m2 (SD = 3.1; range,
30.0–38.9), and a mean percent body fat of 34.4 (SD = 4.2;
range, 26.7–43.5). Lean body mass and percent body fat were determined
by hydrostatic weighing. In part B, 12 nonobese men (BMI, 27.0) were
studied; they had a mean age of 22.2 yr (SD = 2.3; range,
19–27) and a mean BMI of 24.3 kg/m2 (SD = 1.8;
range, 20.8–27.0). All subjects (parts A and B) had unremarkable
histories, physical examinations, and electrocardiograms (ECGs). All
had normal serum chemistries, complete blood counts, TSH levels, and
urinalyses. The mean serum total IGF-I concentration in obese subjects
was 206 µg/L (SD = 66.8; range, 70–309). Subjects were
nonsmokers and had used no medications, alcohol, or caffeine in excess,
nor had they engaged in strenuous exercise for a minimum of 2 weeks
before the initiation of or while enrolled in the study. Subjects were
given ferrous sulfate to take between study days to prevent anemia.
Protocols
Part A. To determine the effects of two doses of L-692–429, 12 obese subjects were entered into a randomized, double blind, placebo-controlled, 3-period cross-over study. On 3 admissions, separated by at least 1 week, fasted obese subjects received a 15-min iv infusion of placebo (saline), low dose L (0.2 mg/kg), or high dose L (0.75 mg/kg). These doses were selected based on previous studies in healthy young men (17). On a fourth (nonrandomized) admission, 6 of the 12 obese subjects received an iv bolus of 1 µg/kg GHRH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)-NH2 (GEREF, Serono Laboratories, Norwell, MA) a dose that has previously been shown to produce maximal GH secretion in healthy young men (21). Subjects were admitted to the GCRC at 2200 h on the evening before the study and fasted from midnight until 4 h after the administration of drug or placebo; water was allowed ad libitum throughout the admission. Subjects assumed a semirecumbent position, and two iv cannulas, one for administration of drug and one for blood sampling, were placed 120 min before study.
Blood samples were obtained every 5 min beginning 1 h before the administration of drug or placebo for measurement of serum GH concentrations, and 15 min before and 0, 15, 60, 120, 240, and 480 min after the initiation of infusion for serum PRL and cortisol levels, the only two pituitary or peripheral hormones shown to be affected by L administration in earlier studies (17). Subjects rested quietly, but were not allowed to sleep. Continuous ECG monitoring and frequent vital signs were obtained for 4 h after treatment. All subjects returned on the morning after each study day and 1 week after completion of the protocol for a laboratory assessment.
Part B. To compare the GH response to low dose L (0.2 mg/kg) in the obese with that in the nonobese subjects as well as to determine the effects of feeding on the response to this dose, nonobese subjects were entered into a double blind, placebo-controlled, randomized, two-period cross-over study. In 10 subjects, on 1 of two admissions, low dose L was administered iv over 15 min after subjects had fasted from midnight (fasted). On the other admission, the same dose was administered 45 min after eating a standardized breakfast (fed). To permit blinded assessment of adverse effects, 2 subjects received placebo (saline) on both admissions. The standardized breakfast was an 817-Cal meal ingested over 15 min while sitting up. It was composed of 14% protein, 28% fat, and 58% carbohydrate.
Blood samples were obtained every 15 min beginning 1 h before the administration of drug or placebo for measurement of serum GH concentrations. Serum PRL and cortisol levels were measured as described in part A and at additional points of -60, -45, and -30 min. Serum glucose was measured at all time points. ECG monitoring and vital signs were obtained in a manner identical to those in part A.
Assays
Routine serum chemistry, hematology, urinalysis, and baseline hormone levels were measured by standard methods at the University of Virginia Clinical Laboratories or the GCRC Core Laboratory (part A) and the Nichols Institute (San Juan Capistrano, CA; part B). Total serum IGF-I was measured by RIA after acid-ethanol extraction (Nichols Institute Diagnostics), with an assay sensitivity of 13.5 µg/L and mean intra- and interassay coefficients of variation of 10.7% and 13.8%, respectively (part A). For hormone assays, all samples from an individual were analyzed in the same assay.
Serum GH concentrations in samples collected every 15 min in all obese and nonobese subjects were measured in duplicate by immunoradiometric assay (IRMA) using standards diluted in human serum (Nichols Institute Diagnostics). The sensitivity of the assay was 0.6 µg/L. The mean intra- and interassay coefficients of variation were 5.9% and 7.8%, respectively. Additionally, in the subset of six obese subjects who also received GHRH, the samples collected every 5 min were assayed in duplicate using the Nichols Luma Tag hGH chemiluminescence assay (Nichols Institute Diagnostics), modified as previously described to enhance assay sensitivity to 0.002 µg/L (22). GH was measurable in all samples using this assay.
Serum cortisol was measured in duplicate by RIA (Nichols Institute Diagnostics); the assay sensitivity was 1.0 µg/dL. The mean intra- and interassay coefficients of variation were 6.8% and 10.8%, respectively. Serum PRL was measured in duplicate by IRMA (Nichols Institute Diagnostics); the assay sensitivity was 0.4 µg/L. The mean intra- and interassay coefficients of variation were 5.3% and 6.1%, respectively.
Data analysis
ANOVA with Duncan’s multiple comparison test was used to test for significant differences among treatments in the 12 obese subjects as well as in the subset of obese subjects who received GHRH (part A). GH concentrations measured with the IRMA that were below 0.6 µg/L were set equal to 0.6 µg/L for analysis. ANOVA for a two-period cross-over design was used to compare responses in the fed and fasted states in the nonobese subjects (part B). Unpaired t tests were used to compare the peak GH responses to low dose L in the obese and nonobese subjects. In the subset of 6 obese subjects, linear regression analysis was used to test for correlations between GH responses and BMI, percent body fat, and baseline total IGF-I concentration. Results are expressed as the mean ± SE except where indicated. Statistical significance was assumed for P < 0.05.
Results
Part A (obese young men)
GH release was significantly stimulated by both doses of
L-692,429. Mean serum GH responses in the 12 obese subjects are shown
in Fig. 1
. Peak GH responses were 0.84 ± 0.1,
12.6 ± 1.8, and 18.5 ± 2.7 µg/L to placebo, 0.2 mg/kg L,
and 0.75 mg/kg L, respectively, (P < 0.05
vs. placebo).
|
.
Age, BMI, and IGF-I before admission are shown for comparison with
individual responses. Regression analysis of these data revealed that
only age (0.75 mg/kg L) and baseline IGF-I levels (both 0.2 and 0.75
mg/kg L) were predictive of GH response. Specifically, increasing age
was associated with a decrease in GH responses, and decreasing IGF-I
levels were associated with a greater GH response. There was no
correlation with either BMI or percent body fat. In addition, in these
six subjects, peak GH responses to both doses of L (17.3 ± 4
µg/L to low dose and 23.4 ± 4.8 µg/L to high dose) were
significantly greater than that to placebo (0.2 ± 0.1 µg/L) and
GHRH (4.0 ± 1.4 µg/L; P < 0.05). Although not
statistically significant, 10 of the 12 obese subjects had numerically
greater peak responses to high dose than to to low dose L, suggesting a
dose-response relationship.
|
). Although
statistically significant, none of the values after the administration
of 0.2 mg/kg L was outside the normal range, and levels of both
hormones returned to baseline by 120 min after the administration of
both low and high dose L.
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Part B (nonobese young men)
Mean serum GH responses to 0.2 mg/kg L in 10 nonobese young men in
both the fed and fasted states are shown in Fig. 4. GH
responses in the fasted state were significantly greater than those in
the fed state (P < 0.05, by paired t test;
data not shown for the two subjects who received placebo). In fact, the
responses to low dose L in the fed state were similar to those of the
obese in the fasted state (Fig. 5).
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Adverse effects
There were no changes in any clinical laboratory values, ECGs, or vital signs with L treatment, and it was well tolerated in both subject groups. Warmth and/or flushing were reported in 50% (n = 6) of the obese subjects with 0.75 mg/kg L and in one obese and one nonobese subject with 0.2 mg/kg L. The only other reported side-effects included transient shortness of breath and decreased appetite in one subject each in part A. No consistent treatment or dose-related incidence could be ascertained.
Discussion
Obesity is a relative GH-deficient (GHD) state, although the mechanism for this phenomenon is not completely known. We have shown that L is an effective GH secretagogue in the setting of obesity, with both the 0.2 and 0.75 mg/kg doses causing a statistically significant increase compared to placebo. Additionally, in a subset of six obese individuals, L was a more effective secretagogue than GHRH. This mean peak response to GHRH is comparable to that reported in other studies (6, 8). Low dose L also stimulated GH release in both the fed and fasted states in nonobese young men; however, feeding attenuated the response. Of note, the mean peak GH response to 0.2 mg/kg L in fasted nonobese individuals was approximately 4-fold higher than that in the fasted obese individuals. Interestingly, this dose caused nearly identical stimulation of GH in fed nonobese and fasted obese subjects. It is not known whether the attenuation of GH secretion in obesity is physiological or pathological; however, the similar peak GH responses seen in fasted obese and fed nonobese individuals suggest that the obese state may alter feedback to the hypothalamic-pituitary system in a manner similar to food ingestion. Evidence exists to suggest that somatostatin tone is increased in obesity (6, 23), leading to inhibition of GH responses to secretagogues.
The mechanisms of action of GHRP and L appear to be identical (17), yet they are not completely understood. GHRP acts via non-GHRH receptors (24) at the level of both the hypothalamus and the pituitary (10, 11, 25). It induces activation of intracellular Ca2+ and protein kinase C and phosphatidylinositol hydrolysis (26), in contrast to GHRH, which activates cAMP (27). Moreover, Adams et al. recently reported that L stimulated GH secretion and activated identical second messenger pathways in human pituitary tumors (28), adding further evidence that GHRP-6 and L generate similar receptor and postreceptor events. Importantly, in in vitro and in vivo settings, both GHRP-6 and L have been shown to act as functional somatostatin antagonists (12, 29), providing a potential explanation for their enhanced GH secretagogue activity in obesity compared to that of GHRH.
The results of our study support previous investigations that demonstrated attenuation of GH secretion in obesity (5, 6, 7, 8, 18). Although there were minor differences in study design between parts A and B (part A subjects underwent blood sampling every 5 min compared to every 15 min in part B), we would have expected this difference to have produced higher, rather than lower, GH levels in the obese subjects due to the potential stress of additional sampling. Others have shown that the increased insulin concentrations seen in obesity may inhibit GH secretion via alteration of IGF-I or IGFBP concentrations (30). Interestingly, Orskov and colleagues (31) recently found that free IGF-I levels in obese individuals are elevated as a consequence of decreased IGFBP-1, leading to enhanced negative feedback of GH secretion. Although not measured in this study, we speculate that free IGF-I concentrations are low in fasted nonobese subjects and high in both fed nonobese and fasted obese individuals. The more robust GH response in the former and a similar attenuated response in the latter two groups could be explained by the negative feedback effects of IGF-I on GH secretion. Of interest, both infusions of recombinant human IGF-I (3) and refeeding with a balanced meal (4) rapidly decrease GH secretion in fasted normal weight young men. The similar time course of GH suppression in these two studies suggests that the effects of nutrition on GH secretion may be mediated by changes in bioavailable quantities of circulating IGF-I. Thus, both obesity and food ingestion may lower GH secretion by IGF-I feedback. IGF-I is known to inhibit GH secretion directly at the level of somatotropes (32, 33) and may also stimulate somatostatin release by the hypothalamus (33), although the latter effect may require the presence of IGF-II (34).
Although the benefits of GH replacement in GHD children are clear and well established, the effects of such replacement in GHD adults are less certain, especially when the potential side-effects of chronic GH administration are considered (35). In recent years, many studies have examined the effects of GH administration to different populations of GHD adults, including the relative GH deficiency of the elderly (36). Although the substantial benefits include improved quality of life and reduction of body fat as well as enhanced exercise performance (37, 38), the current method of administering GH or its secretagogues is both costly and inconvenient. Therefore, the development of a therapeutic compound that could be administered orally has obvious significance. It is not known whether obesity or its negative sequelae could be reversed by an increase in GH secretion. Moreover, data from in vitro experiments demonstrate a clear difference between pulsatile (i.e. physiological) and continuous administration of GH; it is unclear whether long term oral administration of a GH secretagogue will be able to sustain GH release in a pattern that would promote its beneficial effects. The availability of such an agent would allow practical investigation of both possibilities.
In conclusion, L is an effective and generally well tolerated GH secretagogue in obese and nonobese young men. Additional studies are needed to determine whether there are side-effects with long term administration as well as its potential therapeutic application in obesity, a relative GH-deficient state.
Acknowledgments
We gratefully acknowledge the expert technical assistance of Mr. Christopher Womack and Dr. Arthur Weltman in the hydrostatic weighing of subjects in part A. In addition, we thank Ms. Sandra Ware Jackson and the nursing staff of the GCRC and the nursing staff at UMDNJ for their expert assistance, the General Clinical Research Center Core Laboratory for performing the chemiluminescence GH assays, and the University of Virginia Medicine Clinical Laboratory for performing the total IGF-I assays.
Footnotes
1 This work was supported in part by a grant from Merck Research
Laboratories and in addition by NIH Grants DK-32632 (to M.O.T.),
AG-10997–03 (to M.L.H.), and RR-00847 (General Clinical Research
Center and Computerized Data Management and Analysis System
Laboratory). 
Received July 16, 1996.
Revised December 27, 1996.
Accepted January 6, 1997.
References
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