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Original Studies |
Department of Medical Sciences and Advanced Therapies, Section of Endocrinology, University of Ferrara (E.C.d.U., M.R.A., A.R.M., G.T., E.P.), I-44100 Ferrara; and the Department of Pharmacology, University of Milan (S.G.C., A.E.R., E.E.M.), I-20129 Milan, Italy
Address all correspondence and requests for reprints to: Ettore C. degli Uberti, M.D., Department of Medical Sciences and Advanced Therapies, Section of Endocrinology, University of Ferrara, Via Savonarola 9, I-44100 Ferrara, Italy.
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Abstract |
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) GH peak (2 ± 0.7 µg/L) after withdrawal
of SRIH infusion was significantly (P < 0.02)
lower than that in young women (7.3 ± 2 µg/L). In elderly men,
the mean GH peak (2.9 ± 0.6 µg/L) after withdrawal of SRIH
infusion was lower than that in young men (6.3 ± 1.6 µg/L),
although the difference failed to achieve statistical significance.
Baseline insulin-like growth factor I levels were significantly lower
in elderly compared to young subjects in both men and in women. In
women, both age and basal plasma estradiol and testosterone levels
significantly correlated with GH peak after SRIH withdrawal (r
= -0.61, r = 0.61, and r = 0.66, respectively), whereas in
men they did not. These findings are compatible with the view that an
age-related decrease in endogenous GHRH function may contribute to the
defective GH secretion of the elderly. Alterations in plasma
concentrations of sex steroids may have important implications in the
observed changes. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). The
young women were tested in the midfollicular phase of the menstrual
cycle.
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Subjects were admitted to the clinical center at least 1 day before the study. The use of alcohol, tea, and caffeine-containing food was prohibited for 10 days before as well as throughout the study. After an overnight fast, an indwelling iv cannula was inserted in both forearms at 0730 h for separate blood sampling and drug administration. An equilibration period of 1 h was allowed before baseline blood samples were obtained. Subjects remained supine and awake in bed throughout the procedures, which were attended by a nurse and a physician. At 4-day intervals, each subject was tested on two separate occasions in a single blind, randomized manner: 1) iv infusion of SRIH (Stilamin, Serono, Milan, Italy) in 50 mL normal saline at a rate of 9 µg/kg·h over 90 min (0–90 min of the study), and 2) iv infusion of normal saline alone (50 mL saline, from 0–90 min of the study). Blood samples were drawn at -30 and 0 (time of commencement of saline or SRIH infusion) and 30, 60, 90, 105, 120, 135, 150, 165, and 180 min for GH. Baseline plasma levels of IGF-I, estradiol (E), and testosterone (T) in all subjects were also measured at -30 min. Plasma SRIH was measured before and 30, 60, and 90 min after initiation of SRIH infusion.
Blood collection and processing
Blood samples were collected in tubes containing 1.5 µg/L ethylenediamine tetraacetic acid disodium salt (EDTA-2Na) for plasma E, T, GH, and IGF-I and in precooled plastic tubes containing EDTA-2Na (1.5 µg/L) and aprotinin (Trasylol, Bayer, Milan, Italy; 1000 kallikrein inhibitor units/mL) for plasma SRIH. They were immediately chilled, centrifuged at 3000 x g for 15 min at 4 C, and stored at -80 C until assayed. All samples for each subject were processed in duplicate in the same assay. For SRIH RIA, octadecylsilylsilica cartridges (Sep-Pak C18, Waters Associates, Milford, MA) were prepared by washing with 5 mL acetonitrile (Sigma Chemical Co., St. Louis, MO) followed by 5 mL water. Weakly bound plasma components were eluted with 5 mL water followed by 5 mL 0.10% trifluoroacetic acid (Aldrich Chemical Co., Milwaukee, MI). SRIH was eluted with 2.0 mL 80:20 (vol/vol) acetonitrile-0.1% trifluoroacetic acid. The eluate was promptly frozen, lyophilized, and stored at -80 C until subsequent assay. The lyophilized eluates were reconstituted in 2.0 mL phosphate buffer (0.05 mol/L; pH 7.2) containing 0.01 mol/L EDTA and 0.3% BSA. SRIH RIA was performed using radiolabeled SRIH ([3-125I]iodotyrosyl11-Tyr11-somatostatin-14), rabbit antiserum to SRIH (code no. 1611), and standard SRIH (SRIH-14) supplied by Amersham Italia (Milan, Italy). The assay sensitivity was approximately 0.03 µg/L, and the intraassay variation was 8%. To avoid interassay variation, all samples were assayed simultaneously. The MCR of SRIH was calculated from the following equation according to Tait (24): MCR = [infusion rat of SRIH/(steady state level of SRIH - preinfusion level)]. Steady state levels of SRIH were determined 90 min after starting the infusion. GH was measured by immunoradiometric assay with reagents supplied by Nichols Institute Diagnostics (San Juan Capistrano, CA). The lowest detection limit was 0.05 µg/L, with intra- and interassay coefficients of variation of 3.3% and 6.1%, respectively. Plasma IGF-I was determined by RIA, using a commercially available kit (Medgenix Diagnostic, Fleurus, Belgium), after acid-ethanol extraction from EDTA plasma. The inter- and intraassay coefficients of variation were 9.6% and 6.1%, respectively. E and T were determined by RIAs using material supplied by Diagnostic Products Corp. (Los Angeles, CA). The inter- and intraassay coefficients of variation were 7.4% and 5.8% for E and 5.6% and 5.2% for T, respectively.
Statistical analysis
All results are expressed as the mean ± SEM.
To facilitate comparison of the GH secretory profiles after withdrawal
of SRIH infusion, plasma GH responses were expressed either as absolute
values or as peak increment in GH (the peak value detected after
termination of SRIH infusion minus the mean value of the plasma GH
levels obtained during the 90-min period of SRIH infusion in each
subject, i.e. GH peak). The results were compared within
each group and between groups using an ANOVA. Student’s paired and
unpaired tests were used to evaluate individual differences between
means. When applicable, a preliminary logarithmic transformation was
used to satisfy the assumption of a normal distribution of variances.
Correlations between GH peak and age or plasma sex steroid levels
were performed with the linear regression analysis.
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Results |
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shows the mean (±SEM) basal plasma GH, IGF-I,
E, and T concentrations in women and men divided into the two age
groups. Baseline IGF-I levels were significantly lower in elderly
subjects than in young subjects in both men and women. Baseline T
levels were significantly lower in elderly women than in young women.
Baseline E levels were significantly lower in elderly subjects than in
young subjects in both women and men. Baseline E levels were
significantly lower in young men than in young women. BMI was
significantly higher in young men than in young women (Table 1). SRIH
withdrawal in young women (Fig. 1)
resulted in an unequivocal rise in plasma GH levels to a maximum of
5.7 ± 2.2 µg/L at 165 min, with a significant
(P < 0.02) difference vs. the saline
infusion. The mean GH peak (7.3 ± 2 µg/L; range, 1.6–18.9
µg/L) was significantly (P < 0.01) higher than that
observed with saline (0.95 ± 0.4 µg/L). Withdrawal of SRIH
infusion in elderly women (Fig. 1) resulted in a small, but apparent,
increase in the plasma GH level, with a significant difference
vs. the saline infusion at 150 (P < 0.05)
and 180 (P < 0.02) min. The mean GH peak (2
± 0.7 µg/L; range, 0.07–6.3 µg/L) was significantly
(P < 0.05) higher than that observed with saline
(0.02 ± 0.09 µg/L). The plasma GH responses at 130
(P < 0.05), 165 (P < 0.02), and 180
min to SRIH withdrawal and GH peak were significantly lower in
elderly women (P < 0.02) than in young women.
Termination of SRIH infusion in young men (Fig. 2) resulted in an apparent increase in
plasma GH levels to a maximum of 3.1 ± 1.2 µg/L at 120 min,
with a significant (P < 0.02) difference
vs. the saline infusion. The mean GH peak (6.3 ±
1.6 µg/L; range, 0.8–14.8 µg/L) was significantly
(P < 0.05) higher than that observed with saline
(0.12 ± 0.1 µg/L). Termination of SRIH infusion in elderly men
(Fig. 2) resulted in a small increase in plasma GH levels to a maximum
of 2.1 ± 0.8 µg/L at 120 min, with no significant difference
vs. saline infusion. The mean GH peak after
discontinuation of SRIH infusion (2.9 ± 0.6 µg/L; range,
1.4–5.1 µg/L) was significantly (P < 0.01) higher
than that observed with saline (0.33 ± 0.2 µg/L). The plasma GH
response to SRIH withdrawal and the GH peak in elderly men were
lower than those in young men, although the difference failed to
achieve statistical significance (P = 0.15). No
significant sex-related differences in the plasma GH peaks after
SRIH withdrawal were found within both groups of young and elderly
subjects. In women, the GH peak after SRIH withdrawal correlated
negatively with age (r = -0.61; P < 0.02) and
positively with basal plasma E and T levels (r = 0.61;
P < 0.02 and r = 0.66; P < 0.01,
respectively). In men, there was no significant correlation between
GH peak after SRIH withdrawal and age, and plasma sex steroid levels.
No sex- or age-related differences in plasma SRIH levels during SRIH
infusion or in the MCR of SRIH were found. No side-effects were
observed during SRIH infusion.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our findings clearly show that withdrawal of SRIH infusion was capable of inducing a clear-cut increase in plasma GH levels in young subjects. This result is in keeping with earlier reports demonstrating the presence of a post-SRIH rebound release of GH in normal young men (22, 27), but not in patients with isolated GH deficiency (28). The magnitude of the rebound GH rise after SRIH infusion was slightly greater in young women than in young men, although the difference failed to achieve statistical significance. This pattern is reminiscent of the greater GH response to GHRH of young women than young men (10). The higher circulating IGF-I levels and BMI found in men compared to women could contribute to the slight gender-related difference.
An apparent rebound release of GH was still detected after withdrawal
of SRIH infusion in elderly subjects, although it was clearly reduced
compared with that occurring in young subjects. In the elderly women
and men, the GH peaks after SRIH withdrawal were 78.8% and 53.5%
lower than those observed in the young women and men, respectively.
However, this achieved statistical significance only in women, not in
men (P = 0.15), probably due to the small numbers of
elderly men who were investigated.
Based on the evidence that SRIH withdrawal induces a GHRH-mediated rebound release of GH (20, 27, 28), our findings suggest that in the elderly, SRIH withdrawal evokes a smaller release of GHRH by the hypothalamus, and that a reduced activity of GHRH-secreting neurons may indeed underlie the defective GH secretion of aging. Reversibility of the decline in GH secretion in old dogs after clonidine administration (29) and in old dogs (23) and humans after fasting (30), maneuvers reportedly directed at the GHRH neurons, support this view. The age-related differences in the magnitude of the GH rebound response to SRIH withdrawal could be due to the SRIH pharmacokinetics, but as no sex- or age-related differences in plasma SRIH levels during SRIH infusion or in the MCR of the peptide were found, this seems unlikely.
Plasma E and T concentrations were significantly decreased in the elderly compared with those in the young women, whereas plasma E, but not T, levels were significantly lower in the elderly than in the young men. In addition, the GH peak after withdrawal of SRIH infusion significantly correlated with circulating levels of both E and T in women, but not in men. These results suggest that the age-related decline in the hypothalamic activity, as inferred from the GH response to SRIH withdrawal, could be attributable to changes in plasma concentrations of sex steroids that occur with aging. An important modulatory role of the latter, with respect to the influence of age on the neuroendocrine control of the somatotropic axis, is in keeping with the well recognized interplay between sex hormones and the hypothalamic-GH-IGF-I axis (for a review, see Refs. 4 and 31).
Based on these studies, we suggest that the more consistent decline in hypothalamic GHRH activity observed in elderly women may reflect the reduced estrogen-mediated facilitatory effect in the neuroendocrine control of GHRH function. In addition, we cannot exclude that in elderly men the decrease in circulating levels of E may also be instrumental in the altered GH response to withdrawal of SRIH infusion. This view is in keeping with the conclusions of previous studies showing that in both women and men, variations in endogenous E concentrations largely account for sex- and age-related differences in total and pulsatile GH release.
In conclusion, we found a substantial decrease in the magnitude of the rebound GH response to the termination of SRIH infusion in elderly compared to young subjects. This event was more consistent in women, in whom the magnitude of the GH response to SRIH withdrawal correlated negatively with age and positively with basal plasma E and T levels. These findings are compatible with the view that in humans, a reduced activity of GHRH-secreting neurons may be involved in the declining GH secretion that occurs with advancing age and that alterations in plasma concentrations of sex steroids may have important implications in the observed age-related changes in the hypothalamic activity.
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Footnotes |
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Received March 26, 1997.
Revised May 28, 1997.
Accepted June 3, 1997.
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