Vol. 279, Issue 4, R1455-R1466, October 2000
Synergy of L-arginine and growth hormone
(GH)-releasing peptide-2 on GH release: influence of gender
Laurie
Wideman1,2,
Judy Y.
Weltman1,2,
James T.
Patrie3,
C. Y.
Bowers4,
Niki
Shah1,
Shannon
Story1,
Arthur
Weltman1,2,5, and
Johannes D.
Veldhuis1,2,6
Departments of 1 Internal Medicine, 3 Health
Evaluation Sciences, and 5 Human Services, 2 General
Clinical Research Center, and 6 National Science Foundation
Center for Biological Timing, University of Virginia,
Charlottesville, Virginia 22908; and 4 Division of
Endocrinology and Metabolism, Tulane University, New Orleans, Louisiana
70112
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ABSTRACT |
We test the
hypotheses that 1) growth hormone (GH)-releasing peptide-2
(G) synergizes with L-arginine (A), a compound putatively achieving selective somatostatin withdrawal and 2) gender
modulates this synergy on GH secretion. To these ends, 18 young healthy volunteers (9 men and 9 early follicular phase women) each received separate morning intravenous infusions of saline (S) or A (30 g over 30 min) or G (1 µg/kg) or both, in randomly assigned order. Blood was
sampled at 10-min intervals for later chemiluminescence assay of serum
GH concentrations. Analysis of covariance revealed that the
preinjection (basal) serum GH concentrations significantly determined
secretagogue responsiveness and that sex (P = 0.02) and
stimulus type (P < 0.001) determined the slope of this
relationship. Nested ANOVA applied to log-transformed measures of GH
release showed that gender determines 1) basal rates of GH
secretion, 2) the magnitude of the GH secretory response to
A, 3) the rapidity of attaining the GH maximum, and
4) the magnitude or fold (but not absolute) elevation in GH
secretion above preinjection basal, as driven by the combination of A
and G. In contrast, the emergence of the G and A synergy is sex
independent. We conclude that gender modulates key facets of basal and
A/G-stimulated GH secretion in young adults.
male; female; pituitary; somatotropin; regulation; endocrine
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INTRODUCTION |
GENDER
DIFFERENCES IN BASAL (unstimulated) pulsatile growth hormone (GH)
secretion are recognized in experimental animals and throughout the
human lifetime (24, 28). Recent clinical studies have
revealed other distinctions in the neuroendocrine control of GH release
in men and women (55). For instance, Pincus et al.
(40) reported that men exhibit more orderly patterns of GH
release than women. Analogous gender differences are evident in the
patterns of GH release in rats (28, 36). Further
laboratory investigations have demonstrated differential regulation of
hypothalamic GH-releasing hormone (GHRH) and somatostatin gene
expression in male and female rodents (4). Distinguishable
mechanisms of GH release in men and women thus are likely to embody
sex-specific neuromodulatory inputs to the hypothalamus and/or
pituitary gland. Gender differences also exist in GH responses to
certain discrete physiological and pharmacological stimuli (17,
27). However, there is only a limited understanding of the exact
neuroendocrine mechanisms that drive differential GH release in men and
women (53).
L-Arginine (A) is a widely employed provocative test to
evaluate the GH axis. Although the mechanism by which A releases GH in
the human is not known definitively, on the basis of some, but not all
studies, in the rat, a current proposition is that A stimulates GH
release via withdrawal of hypothalamic somatostatin (2, 22,
23). Early studies by Merimee et al. (34) reported that women manifest greater GH release in response to A stimulation than men and that this difference is due to estrogen exposure. Penelva
et al. (37) reported that pyridostigmine, which also increases GH release at least in part via somatostatin withdrawal, slightly enhanced the GH secretory response to GH-related peptide (GHRP)-6. GHRP-2 (G) is a novel potent non-GHRH peptidyl stimulus for
GH release in both men and women (7). Whereas some studies have suggested gender differences in GH responses to GHRP, others have
not (6, 9, 37). One recent analysis suggested that there
is no gender variation in maximal GHRP-stimulated GH release but rather
a heightened sensitivity in women to low G doses (7).
In vivo in the rat and human, combined infusions of GHRH and GHRP exert
synergistic rather than additive actions. Whereas the precise
mechanistic basis for this synergism has not been elucidated, the two
effectors clearly activate different cellular signaling pathways
(1, 14). Moreover, each of these peptidyl agonists can
partially restrain the inhibitory effects of somatostatin (24).
The current clinical study investigates the ability of A and G to act
synergistically in healthy young adults and tests the hypothesis that
the synergy is gender dependent.
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METHODS |
Clinical protocol.
Eighteen healthy subjects [9 men (means ± SE) (age = 25 ± 1.5 yr, height = 178 ± 1.0 cm, wt = 75.1 ± 1.8 kg, total body fat = 14.5 ± 1.9%) and 9 women (age = 25 ± 1.0 yr, height = 169 ± 2.0 cm,
wt = 66.5 ± 3.1 kg, total body fat = 22.6 ± 1.8%)] participated in the present study. All volunteers underwent a
detailed screening medical history and complete physical examination
and provided written informed consent as approved by the Human
Investigation Committee at the University of Virginia. Subjects were
not taking any medications or hormones and were moderate habitual
exercisers (20-30 min of aerobic exercise, 3 or 4 times/wk).
Body density was measured by hydrostatic weighing (30).
Residual lung volume was measured by oxygen dilution (62).
Each subject was weighed in air on an Accu-weigh beam scale accurate to
0.1 kg and again underwater on a Chatillon autopsy scale accurate to
10 g. Percentage body fat was calculated using the equation of
Brozek et al. (11).
Subjects were admitted overnight to the General Clinical Research
Center on four separate occasions (at rest). Admissions were scheduled
at least 2 days apart and followed the study-specific randomization
schedule based on gender, which was produced by SAS (Proc Plan). Women
were studied during the early follicular phase (days
2-8) of the menstrual cycle. Volunteers received a standardized constant meal, based on body weight, at 1700 the evening
before the study. Caloric content of the meal was calculated as
0.33 × 37 kcal/kg for females and 0.33 × 38.5 kcal/kg for
males, which included an activity factor for moderate activity
(19). The nutrient composition of the meal was fixed at
55% carbohydrate, 30% fat, and 15% protein. After volunteers fasted
overnight, venous cannulas were placed in contralateral forearm veins
at 0500 and blood samples were withdrawn at 10-min intervals from 0600 to 1200. At 0600 on each admission, blood samples were obtained for later measurements of serum insulin-like growth factor (IGF)-1, total
and free testosterone, and estradiol concentrations. At 0730, an
intravenous infusion of either A (30 g in 300 ml) or saline (S; 300 ml)
was given over 30 min. At 0800, an intravenous bolus of either G (1 µg/kg) or S was given. Subjects rested quietly in their rooms during
the studies.
Assays.
GH concentrations in all serum samples (0600-1200) were measured
using a recently validated ultrasensitive (0.005 µg/l threshold) chemiluminescence-based assay (Nichols, San Juan Capistrano, CA) (13, 26, 58). The chemiluminescent assay detects
predominately the 22-kDa form of GH, with 34% cross-reactivity for
20-kDa GH (methionylated). The median intra-assay coefficient of
variation (CV) for the GH assay was 6.0%, and the interassay CV was
9.9%. Total testosterone, free testosterone (analog assay), and
estradiol concentrations were measured by solid-phase RIA
(Coat-a-Count, Diagnostic Products, Los Angeles, CA). The intra-assay
CVs were 6.9, 3.8, and 3.9% for total testosterone, free testosterone
and estradiol, respectively, whereas the interassay CVs were 10.3, 4.2, and 9.5%, respectively. Serum total IGF-1 was measured by RIA
(Nichols). The intra-assay CV for IGF-1 was 6.7%, and the interassay
CV was 13.6%.
Deconvolution analysis.
A multiple-parameter deconvolution method was used to estimate
pulsatile attributes of GH secretion from the measured serum GH
concentrations (56). A pulse of underlying GH secretion
was approximated algebraically by a Guassian distribution of secretory rates (54). Basal secretion (time invariant) was estimated
concurrently with a subject-specific monoexponential half-life of
endogenous GH. The stepwise procedure for deconvolution entails
prefitting via an automated waveform-independent technique (PULSE2), in
which regions containing significant secretion impulses of undefined waveform are identified successively within a time series when they
significantly reduce the total fitted variance by F ratio testing (29). Peak locations from PULSE2 were used as
estimates in the multiparameter deconvolution analysis, as previously
described (20, 58). To avoid overdetermination of peaks
(Nyquist concept), putative successive GH peaks separated by <20 min
(2 sampling intervals apart) were eliminated and the data were refit.
In addition, any presumptive peaks that were outside the sampling
window (0-370 min) by more than one sampling interval (10 min)
were eliminated.
GH secretory pulses were considered significant if the fitted amplitude
(maximal value attained within the computed secretory event) could be
distinguished from zero with 95% statistical confidence. The GH
secretory pulse half-duration, defined as the duration in minutes of
the calculated secretory burst at half-maximal amplitude, GH half-life
of elimination, and GH distribution volume were assumed to be constant
throughout any one study period in any individual. The mass of GH
secreted per pulse was estimated as the area of the calculated
secretory pulse (µg/l distribution volume). The endogenous pulsatile
GH production rate was defined as the product of the number of GH
secretory pulses and the mean mass of GH secreted per pulse during
6 h. Additionally, the 90-min GH secretory burst mass, defined
here as the total mass of GH secreted in 90 min from the end of
infusions (0800-0930). This measure limits the effect of spurious
spontaneous GH release at later times in the sampling session.
Statistical procedures.
Between-group comparisons were expressed in terms of fold change in the
value of the geometric mean (GM). The GM is a location parameter,
similar to the arithmetic mean and median, and is calculated by simply
taking the antilogarithm of the mean response computed from the
logarithmically transformed data. We compared GMs instead of arithmetic
means, because one of the critical statistical model assumptions for
ANOVA states that, to obtain valid statistical tests, residual
variation should be approximately equal within all treatment groups.
When the magnitude of the variance in the response increases as the
mean of the response increases in value, the natural logarithmic
transformation is generally used to stabilize the residual variance
among two or more treatment groups (18). Therefore, data
for serum sex steroids, IGF-1, integrated GH [area under the curve
(AUC)], and calculated GH secretion parameters were transformed to the
natural logarithm scale. Total testosterone was not logarithmically transformed.
All ANOVA computations were carried out in SAS version 6.12 (SAS/STAT
Software Changes and Enhancements, 1996), with the mixed model software
of Proc Mixed. Parameter values were estimated by restricted maximum
likelihood (REML), and nonexact F tests were approximated by
using a Satterthwaite approximation (31). A Bonferroni
multiplier with a prespecified experimental type I error rate of 0.05 was used to adjust probabilities and confidence limits to maintain a
type I error rate of 0.05 for all comparisons of interest. All
P values presented correspond to statistical tests that were
conducted on the log-transformed response data.
Total (integrated) AUC for the serum GH concentration-response
curve was computed using the trapezoidal rule (32). These and other GH response estimates were then analyzed by a three-way nested ANOVA model, with gender, condition, and stimulus type considered classification variables.
An analysis of covariance (ANCOVA) model was used to determine whether
the values of basal preinjection GH (measured just before each
stimulus) predicted subsequent GH release. As a component of the ANCOVA
model, the basal GH level was treated as a continuous covariate.
Classification variables for gender, condition, and stimulation were
also included in the ANCOVA model as well as terms for two-way,
three-way, and four-way interactions among the values of basal GH and
the classification variables. Parameter values were estimated by REML
(see above).
A test of nonadditivity was used to assess the a priori hypothesis that
GH release induced by combined G and A infusions is independent or
synergistic. In this analysis, we eliminated the baseline intervals of
GH release before and after the stimulation period, which is thus
defined as 0730-1030. A formal derivation of this statistical test
for nonadditivity is included in the APPENDIX. Note that in
this derivation the value of the GH response of each subject is
calculated by simply adding his/her baseline-adjusted estimates of
serum GH AUC after stimulation by A and stimulation by G and then
subtracting this sum from the estimate of baseline-adjusted serum GH
AUC after combined AG stimulation. This analysis thus tests the null
hypothesis of no synergy (independence) between the GH-stimulatory
effects of A and G delivered together (AG). This procedure was repeated
for 90-min GH secretory burst mass. Nonadditivity terms were analyzed
by a two-way nested ANOVA model, with gender and condition treated as
classification variables.
To assess power for the current investigation, values of minimum
detectable fold change in the GM were estimated for the primary outcomes [stimulated serum GH integrated area (AUC) and 90-min secretory burst mass] on the basis of the three-way nested ANOVA that
was used in the analysis (45). In the computations of
minimum detectable fold-change, the power of the statistical test was specified to be 0.80 and the type I error rate was specified to be
0.05. For comparisons of GH AUC and 90-min burst mass that involve
two-way interactions the estimates of the minimum detectable fold
change were 1.23 and 1.56, respectively, whereas the estimates were
1.47 and 2.26, respectively, for three-way interactions.
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RESULTS |
The group serum GH concentration response patterns over time to
each of the stimuli are shown in Fig.
1A for men and Fig. 1B for women. The maximal serum GH concentration attained
was greatest for the AG stimulus and least for S. The rank order of stimulus strength, AG > G > A > S, was the same in
men and women. In men, the values of the maximal serum GH
concentrations attained after each of the stimuli were 2.4, 7.8, 36, and 73 µg/l (for S, A, G, and AG, respectively). These data show a
30-fold increase in the maximal serum GH concentration in response to
AG compared with S in men. In women, the values for the S, A, G, and AG
treatments were 6.1, 22, 43, and 93 µg/l and the increase in the
maximal serum GH concentration for the AG compared with the S stimulus was 15-fold. Comparing the fold changes between women and men for each
of the stimuli revealed a significant 2.8-fold [95% constant load
(CL) (1.1,7.2)] greater GH rise in response to A in women compared
with men.
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Fig. 1.
Mean serum growth hormone (GH) concentration profiles basally and
in response to GH-releasing peptide (GHRP)-2 (G) and/or
L-arginine (A) infusions in men (A) and women
(B). Data are the means ± SE. Clock time is shown.
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In women, the time to reach the maximal serum GH concentration was
greatest after the A and AG stimuli (50 and 60 min) compared with G (30 min). The same pattern of responses was observed in men, but the time
to reach the maximal GH concentration was greater (delayed) for all
three stimuli (60, 70, 40 min for A, AG, G, respectively). This
consistent gender difference in median time to reach maximal GH
concentration was highly significant (P < 0.001).
Representative individual serum GH concentration vs. time curves for a
man and a woman are shown in Fig.
2A, and the corresponding calculated GH secretion profiles assessed by deconvolution analysis are
given in Fig. 2B (discussed below).
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Fig. 2.
Representative serum GH concentration profiles
(A) and the corresponding GH secretion profiles
(B) in an individual healthy young man and woman basally and
in response to G and/or A infusions. Time zero corresponds
to 0600 clock time in Fig. 1.
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Figure 3 presents box plots, which
summarize the data for total serum integrated GH AUC (logarithmic
scale), for men and women after each of the stimuli. Akin to maximal
serum GH levels, the order of AUC response magnitudes was AG > G > A > S for both men and women. The increase in serum GH
AUC for the AG compared with the S stimulus was 24-fold for men and
11-fold for women. The incremental change in serum GH AUC observed for
each stimulus was influenced significantly by gender (P < 0.001). After A infusion, women had a 3.3-fold [95% CL(1.2,9.2)]
greater median serum GH AUC than men (P < 0.001). No
gender differences existed for the S, G, and AG stimuli.
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Fig. 3.
Box plot representations of log [integrated serum GH area under
curve (AUC)] basally and in response to G and/or A infusions in
healthy young men and women.  , Measurements below the
10th or above the 90th percentile.
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Table 1 presents data from the
deconvolution analysis of GH secretory responses. The basal GH
secretion rate was greater in women than men (P = 0.05), and this difference was consistent regardless of the stimulus
administered. The calculated GH half-life was dependent on the stimulus
administered (P < 0.001), but independent of gender
(P = 0.9). The 90-min GH secretory burst mass showed a
graded stimulus order, which was the same in men and women (AG > G > A > S). The increase in 90-min GH secretory burst mass
after AG infusion compared with S was 242-fold for men and 40-fold for women. The incremental change in 90-min GH secretory burst mass for
each stimuli was significantly influenced by gender (P = 0.005). In the control (S) session, the 90-min secretory burst mass
was greater in women than men (4.8 vs. 0.88 µg/l, P = 0.0003). Women tended to exhibit greater 90-min GH mass after A
infusion (P = 0.072). For both the G and AG stimuli,
men and women had similar 90-min GH mass.
The order of response magnitude for endogenous GH production rate
(AG > G > A > S) was identical to that of 90-min GH
mass (above) for both men and women. The increase in the endogenous GH
production rate from the S baseline to AG stimulus was 23-fold in men
and 7-fold in women. The incremental change in endogenous GH production
rate from one stimulus to the next was influenced by gender
(P < 0.001). In the S and A treatments, women had
greater endogenous GH production rates than men (P < 0.001 and P < 0.001, respectively). Gender differences
were not observed for the G and AG stimuli.
There was a main effect of stimulus on mass of GH secreted per burst
(P = 0.0001), and the gradation of the effect was
AG > G > A > S (data not shown). The mass of GH
secreted per burst in the combined groups increased by 3.5-fold in
response to A infusion over S [95% CL(2.7,5.4)], by 1.7-fold for G
over A stimulation [95% CL(1.1,2.5)], and by 1.8-fold for AG over G
stimulation [95% CL(1.2,2.7)] (P < 0.001 for each).
The mean GH secretory burst amplitude was greater in women than men for
the A and S stimuli (P < 0.001 for both). Women had a
2.9-fold [95% CL(1.2,7.2)] greater mean GH secretory burst amplitude
compared with men after the A stimulus and a 3.0-fold [95%
CL(1.2,7.5)] greater mean GH secretory burst amplitude in the S
(control) setting.
Figure 4 shows a box plot of the results
for the test of nonadditivity applied to the 90-min GH secretory burst
mass. This analysis revealed a synergistic effect for the combined AG
stimulus compared with the individual effects of A and G
(P = 0.02). We noted that the combined AG stimulus
compared with the summed response for (A + G) for 90-min GH
secretory burst mass was 1.3-fold greater in women [95% CL(0.97,
1.8)] and 1.48-fold in men [95% CL(1.1,2.0)].
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Fig. 4.
Box plot representations of the test of nonadditivity of the
logarithms of the 90-min GH secretory burst mass after combined A and G
infusions (AG) vs. their algebraically summed individual effects
(A + G). , Measurements below the 10th or above
the 90th percentile.
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Figure 5 shows a box plot of the
results for the test of nonadditivity for stimulated GH AUC. After
adjustment for baseline (treatment effect at rest), the test for
nonadditivity also revealed that the individual effects of A and G in
determining GH release are not simply additive, but rather
significantly synergistic (supra-additive) when administered in
combination (joint stimulus, AG) (P = 0.003). The
combined AG stimulus compared with summed response for (A + G) was
1.6-fold greater in women [95% CL(1.2,2.0)] and 1.6-fold in men
[95% CL(1.2,2.0)].
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Fig. 5.
Box plot representations of the test for nonadditivity of the
logarithms of the stimulated serum GH AUC after combined AG infusions
vs. their algebraically summed individual effects (A + G).
, Measurements below the 10th or above the 90th
percentile.
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There were no significant gender or stimulus differences in serum
estradiol concentrations or serum IGF-1 concentrations. Men had
significantly greater total and free testosterone concentrations (P < 0.001, for both), independent of stimulus.
ANCOVA revealed that the linear relationship between log (basal serum
GH AUC) and log (stimulated GH AUC) varied with gender (P = 0.02) and stimulus type (P < 0.001). The relationship between these two variables had a positive
slope for the S, A, and AG stimuli, but not for G (results not shown).
When considering all possible interactions between the log (basal serum
GH AUC) and the indicator variables for gender and stimulus type,
ANCOVA showed that including log (basal serum GH AUC) explains
substantial residual variance otherwise unaccounted for by gender and
stimulus (P < 0.001). However, the additional
covariate [log (basal serum GH AUC)] failed to change the composition
of the terms that were judged to be important in the original ANOVA model.
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DISCUSSION |
A major observation in the current study is that the effect on GH
release of A and G administered together is not additive but rather is
synergistic and that such synergism is expressed equivalently in men
and women. Second, a gender-independent rank order of maximal GH
secretory responses is demonstrated, i.e., AG > G > A > S. The synergistic interaction between G and A evoked a massive
outpouring of GH over the succeeding 90 min; namely, a 242-fold (men)
and a 40-fold (women) augmentation of GH secretory burst mass over the
saline control. Third, mean integrated and maximal serum GH
concentrations, the (90 min) mass of GH secreted, GH secretory burst
amplitude, and the GH production rate after A infusion are all higher
in women than men. Last, a further gender difference was identified in
that men had a consistent delay in attaining maximal serum GH
concentrations compared with women. In contrast, the GH half-life,
burst duration, burst frequency, and GH secretory responses to G
infusions are sex independent.
The GH secretory response patterns to each of the distinct
secretagogues were similar qualitatively in men and women (Fig. 1,
A and B). Although the absolute value of the
maximal serum GH concentration attained during the combined AG stimulus
was greater in women than in men (93 vs. 73 µg/l), this difference was not statistically significant. However, given the gender disparity in basal (prestimulus) serum GH concentrations, we also calculated the
fractional (fold) GH increase over basal after combined AG infusions
compared with the unstimulated admission (S). This fold effect
of AG was greater in men (30-fold) than women (15-fold). The augmented
fractional secretory response (despite similar maximal serum GH
concentrations) in men reflected the lower mean serum GH concentrations
in the unstimulated state (S), as recognized in other gender
comparisons (17, 52). Thus, by the way of simple
integrated serum GH responses, men evinced a 24-fold and women an
11-fold rise above control (S) under joint AG drive (P < 0.001). The possible physiological role that this difference may
play in mediating gender differences in target organs of GH action
(i.e., body mass and structure) is unknown.
The time to reach the maximal serum GH concentration was greater after
the A and AG stimuli compared with G in both genders. For each
secretagogue, men attained a maximal serum GH concentration ~10 min
later than women (in A, G, and AG). This temporal disparity is similar
to that observed in a recent study using exercise as an alternative
stimulus (63). Deconvolution analysis showed that the
delay was not due to postponed GH secretion, a more prolonged GH
half-life, or an extended GH secretory burst in men. There are several
possible mechanistic explanations for this gender distinction. First,
men's lower basal serum GH levels may have extended their time to
reach an equivalent maximal GH peak. Second, procedural factors such as
the introduction of intravenous fluids (S, A, or G) may have elicited
unequal stress responses in the two sexes, given that
corticotropin-releasing hormone (CRH) release (at least in the
rat) evokes somatostatin secretion (38). In addition, the
sexually dimorphic patterns of GH secretion are likely controlled at
hypothalamic loci, as inferred from laboratory studies in rats
(47, 48, 49). In the human, Jaffe et al. (27)
also reported a gender-dependent pattern of IGF-1 feedback control of
GH secretion, in which women exhibit reduced inhibitory responsiveness
to IGF-1 infusions, possibly reflecting a lesser effect of (daytime)
somatostatin in women. Earlier, Carlsson et al. (12)
described sexually dimorphic GH autofeedback control of GHRH and
somatostatin actions in the adult rodent. Thus we reason that if women
have diminished GH and/or IGF-1 (auto) negative feedback compared with
men, then peak GH release may occur more rapidly in women than men. In
this regard, application of the approximate entropy statistic, taken as
an indirect barometer of within-axis feedback signaling, at the
hypothalamic-pituitary level, strongly distinguished the orderliness of
GH release in the female and male in both human and rat
(40). The latter strongly suggests (but does not prove)
unequal GH-IGF-1 feedback strength in the two sexes (60).
As first described by Merimee et al. (34) over 30 years
ago, we also observed that women attained a significantly (2.8-fold) greater maximal serum GH concentration and (3.3-fold) greater AUC
during L-arginine infusion than men. By deconvolution
analysis, we could corroborate this sex discrepancy and further
explicate mechanistically for the first time that higher maximal serum
GH concentrations during A infusion in women do not reflect any
gender-dependent differences in GH half-life or GH secretory burst
duration or women's higher interpulse serum GH concentrations, but
rather is due to a specific amplification of GH secretory burst mass in
the female. The gender difference in women's heightened GH response to
A is likely estrogen dependent, because men also show elevated
A-stimulated GH secretion after short-term estrogen exposure (34). However, the early follicular phase women studied
here maintained serum estradiol concentrations that were no different from those in the normal men and still manifested accentuated GH
release. Thus an inhibitory effect of testosterone on the GH secretory
response to L-arginine is a possible explanation, as suggested indirectly by other earlier studies of spontaneous (basal) 24-h GH release in men treated with nonaromatizable androgen
(59) or an androgen-receptor antagonist (35).
This putative inhibitory effect of androgen could be mediated via
augmented somatostatin release, as suggested in the rat
(3), and/or increased responsiveness of the pituitary
gland to somatostatin. Although clinical data relevant to the latter
conjecture are not available to our knowledge. Recent clinical
experiments indicate that estrogen does modulate somatostatin action in
women (10). Gender differences after A are distinct and
evidently do not extend to the putative endogenous GHRP-ligand
activated pathway, because at least at the G dose of 1 µg/kg used
here and in earlier studies, men and women exhibit similar
GHRP-stimulated GH release (present data and Refs. 6, 37).
A recent clinical investigation of gender differences in GH release
noted 68-fold higher morning ambulatory serum GH concentrations determined by immunofluorometry (IFA) in young women compared with
young men (17). As observed earlier by immunoradiometric assay and IFA or 24-h GH profiles (52), daily GH secretion
rates are greater in young women than men. We show here that this
gender difference is also present as assessed by ultrasensitive
chemiluminescence-based assay. Furthermore, by deconvolution analysis
we could observe that the sex difference is accounted for solely by
augmented GH secretory burst mass, but not half-life, in women.
Although the calculated (endogenous) GH half-life rose after combined
AG infusions (compared with the individual secretagogues), this effect
was unrelated to gender. Sex invariance of GH half-life also was
evident in experiments using recombinant human GH infusions in
octreotide-suppressed young women and men (44). The
increase in estimated GH half-life observed here after joint AG
administration thus is more likely due to the higher serum GH
concentration attained in this context, because several investigations
have reported a concentration dependence of metabolic clearance rate of
GH in humans (25, 43, 44).
In the current analysis, we calculated the mass of GH secreted over the
entire 6-h study and also evaluated the mass of GH secreted over the
90-min poststimulus interval to obviate the effects of spurious
spontaneous GH release at later times in the sampling session. The rank
order of stimulus effects (AG > G > A > S) was the
same for both measures. In both analyses, women maintained greater
values in both the control (S) and A sessions compared with men, with
no gender difference after G or combined AG infusions.
Regulation of GH secretory burst amplitude explained unstimulated
gender differences as well as the stimulatory effects of A, G, and AG
infusions in this study. Thus the median GH secretory burst amplitude
was greater in women compared with men both under control conditions
and in response to A stimulation, but not after the G and AG stimuli.
On the basis of analytic considerations (57), GH secretory
burst amplitude thus also explicates the gradation of secretagogue
effects (AG > G > A > S). Secretagogue drive of GH
secretory burst amplitude is here shown to be highly specific, given
the failure of any single stimulus to alter GH secretory burst
duration, frequency, or half-life, which are the other (analytic)
determinants of the serum GH concentration (54, 56, 57).
There were no gender differences in serum IGF-1 or estradiol
concentrations in this study, given that women were evaluated during
the early follicular phase of the menstrual cycle. As expected, men
maintained significantly greater total and free serum testosterone concentrations. Of interest, no sex steroid measures correlated with GH
secretory responses to any of the secretagogue stimulus combinations
used here.
Available clinical studies report considerable variability in GH
secretory responses to virtually all stimuli, e.g., exercise, GHRH
challenge, etc. (42, 50, 61). This biological variability in the GH response has led investigators to examine the statistical power of studies with small subject numbers (such as the current study
with 18 subjects), particularly when two- or three-way interactions are
investigated. To this end, we performed power analysis for the primary
GH outcome variables (stimulated GH AUC and 90-min GH burst mass) to
estimate the minimal detectable difference required for two- and
three-way interactions. For these primary outcome variables, the fold
changes observed in the present investigation (range 3.3- to 242-fold)
were much larger than the minimum detectable fold change values (range
1.23- to 2.26-fold), thus ensuring our ability to detect differences in
the GH response despite the large biological variability. Devesa et al.
(16) suggested further that the magnitude of GH release
varies depending on the timing of GHRH infusion, i.e., whether GHRH is
given during a trough or a peak of GH release, reflecting presumptively
high or low somatostatinergic tone, respectively. Given this predicate,
we also evaluated how the prestimulus serum GH concentration influenced the calculated GH secretory response in relation to gender and secretagogue type. This analysis revealed positive relationships between log(basal serum GH integrated AUC concentrations) and log(stimulated AUC serum GH concentrations) at baseline and in response
to the A and AG stimuli, but not the G stimulus. This new observation
for these secretagogues has several implications. First, because the
linear relationship between these two variables depends on the
secretagogue administered, it would become inappropriate to use an
ANCOVA with log(basal GH) as a covariate for the G stimulus if the
intent is to adjust treatment means (46). Second, the evident lack of any significant relationship between basal serum GH
level and G-stimulated GH release warrants further mechanistic evaluation, especially because no (or a negative) correlation existed
in both men and women. As such, the G stimulus would appear to differ
mechanistically from A and GHRH actions (16). Anatomic evidence for interconnections between GHRH and somatostatin neurons in
the hypothalamus (5, 33) might provide one mechanism for this observation. For example, an elevated basal serum GH concentration before G administration might impose greater GH autofeedback on endogenous GHRH (and elevated feedforward on somatostatin) release and
thereby result in reduced (stimulated) GH secretion. This scenario
would not prevail equally in the face of A or AG infusions, because A
is believed to restrain somatostatin release and somatostatin is
thought to mediate GH autofeedback (24). Last, our
exploration of an ANCOVA model demonstrates that significant residual
variance in selective secretagogue-stimulated GH secretion is indeed
explicable by basal GH levels, even beyond the influence of gender.
The mechanism underlying A-induced GH release is currently believed to
include somatostatin withdrawal (2, 21, 22). Although this
mechanism has not been confirmed directly in humans, A has no known
direct effects on GHRH release or the GHRP effector pathway. Current
evidence supports the view that G acts via novel, non-GHRH and
non-somatostatin receptor mechanisms. Therefore, we predicted that the
impact of combined AG stimulation on GH release would exceed that of
either A or G alone, or their algebraically additive effects; i.e.,
represent a supra-additive (or synergistic) interaction. We here
support this important postulate in both men and women. To this end, we
statistically evaluated the null hypothesis that GH release produced by
combined AG stimulation exceeds that of the summed effects of A and G
given on different days. Several previous clinical studies have
reported synergistic (supra-additive) interactions between GHRP and
GHRH (but A was not evaluated) on GH release in humans (6, 8, 9,
39, 41), although another study did not (51). A
synergistic interaction between GHRH and GHRP cannot be accounted for
readily via stimulation of endogenous GHRH secretion or by inhibition
of somatostatin release (6, 41). Alternative hypothetical
mechanisms thus include inhibition of somatostatin actions at the
pituitary level or release of another unknown hypothalamic factor ("U
factor"). In other studies, pyridostigmine [which also presumptively
acts in part via hypothalamic somatostatin withdrawal and in part via GHRH release (24)] augmented the GH secretory
response to GHRP-6 (37), but did not enhance the amount of
GH secreted in response to GHRH given with GHRP-6 (15).
The authors concluded that somatotrope secretory responsiveness to the
combined administration of GHRH and GHRP-6 is largely independent of
somatostatinergic tone (15). However, one must also
consider the possibility that maximal GH release was attained after
combined GHRH and GHRP-6 infusions and that the addition of
pyridostigmine (or any other stimulus) could not enhance GH secretion
further. The present study indicates clearly that combined AG
stimulation is synergistic in both men and women, i.e., greater than
the summed effects of individual infusions of A and G
(P < 0.001). To our knowledge, this is the first study
to show a synergistic impact of A and G on GH release. In addition, we
demonstrate that the effect of combined AG is gender independent
(P = 0.28).
Because the combined AG stimulus response exceeded GH release driven by
the summed A plus G responses in both men and women, we speculate that
A and G might interact in some excitatory manner so as to increase net
GH secretion. Several hypotheses could explain the foregoing
observation. Although considered a minor action of GHRP
(7), partial attenuation of somatostatin action at the
pituitary level by GHRP and/or release of another unknown hypothalamic
factor by GHRP may be responsible for the synergistic action of A and
G. Additionally, inferred bidirectional neuronal connections between
GHRH, somatostatin, and/or endogenous GHRP-like neurons within the
hypothalamus may increase net GH release during the combined infusion
of A and G, i.e., GHRH neurons, which are known to be activated by GHRP
in experimental animals (24), may also stimulate
somatostatin withdrawal. Thereby, the joint AG effect could be greater
than the summed effect of A and G given on separate days. Accordingly,
further analysis of the mechanisms underlying the distinctly
synergistic effects of A and G will likely be fruitful in future
clinical and experimental investigations in both sexes.
Perspectives
The basis for the sexually dimorphic patterns of GH secretion in
humans is a complex issue. Indeed, isolation of factors contributing to
gender difference is made challenging by the inability to appraise directly in vivo each of the neuroregulatory pathways involved. The
present study uses two selective neuroregulatory probes to explore the
mechanisms underlying the sexually dimorphic pattern of GH secretion.
Thereby, we observe both common and sex-specific features of GH
control. First, a common effect of secretagogues on GH release is to
amplify GH secretory burst mass in both men and women. This mechanism
may be important, because data in experimental animals indicate that
increased GH secretory burst mass and elevated basal GH secretion
impact intermediary metabolism, hepatic gene expression, body fat
deposition, and lean muscle mass differentially. Second, these analyses
unveil several gender-specific regulatory features, including basal GH
secretion and individual vs. synergistic secretagogue actions (see
abstract). Finally, the prestimulus basal GH secretion rate influences
the response to secretagogues beyond that explicated by gender alone.
The predictive power of presecretagogue GH levels on responsiveness to
A but not G thus is both gender specific and secretagogue dependent.
The latter may be due to the unequal impact of the concurrent level of
somatostatinergic activity on responsiveness to different specific
secretagogue types. According to this reasoning, the response to G in
either men or women could be largely independent of somatostatin tone. These elements of GH neuroregulation highlight the need for further investigations of gender differences in the multisecretagogue control
of the GH-IGF-1 axis.
| |
APPENDIX |
Test of Additivity
Model statement
where µ is the grand mean; 
i, the
ith gender effect, assumed to be fixed;
j(i) is the
jth subject effect nested within the ith gender,
assumed to be random; 
k is the kth
treatment effect, assumed to be fixed; 
l is
the lth condition effect, assumed to be fixed;
()ik is the effect of the ith
gender by kth treatment interaction; ()il is the effect of the ith
gender by lth condition interaction;
()kl is the effect of the kth
treatment by lth condition interaction;
()ikl is the effect of the
ith gender by kth treatment by lth
condition interaction; and
j(i)kl is
the random error.
Model constraints.
Test of additivity.
Let *1 be the baseline adjusted treatment effect
of A, *2 be the baseline adjusted treatment
effect of G, and *3 be the baseline adjusted
treatment effect of combined A and G (AG).
Note that
Thus for the new response
y*j(i)l = y(AG 
A
G)j(i)l, the model can be expressed as
where
Hence
and the null hypothesis for a global test of additivity is that
µ*= 0.
| |
ACKNOWLEDGEMENTS |
The authors acknowledge the invaluable contributions of the
following individuals to the present project: Sandra Jackson and the
nurses in the General Clinical Research Center for drawing blood and
caring for patients and Ginger Bauler, Katherine Kern, and Eli Casarez
for performing the GH chemiluminescence and other radioimmunoassays.
| |
FOOTNOTES |
This study was supported in part by General Clinical Research Center
Grant RR-00847, the National Science Foundation Center for Biological
Timing, and National Institutes of Health Grant R01-AG-147991 to J. Veldhuis.
Address for reprint requests and other correspondence: J. D. Veldhuis, Division of Endocrinology and Metabolism, Dept. of Internal Medicine, School of Medicine, Univ. of Virginia,
Charlottesville, VA 22908 (E-mail: jdv{at}virginia.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 1999; accepted in final form 22 May 2000.
| |
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ABSTRACT
INTRODUCTION
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