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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 11 5160-5167
Copyright © 2002 by The Endocrine Society

Original Article

Aging-Related Changes in Release of Growth Hormone and Luteinizing Hormone in Female Rhesus Monkeys

Michael J. Woller, Gina Everson-Binotto, Elana Nichols, Ashley Acheson, Kim L. Keen, Cyril Y. Bowers and Ei Terasawa

Wisconsin Primate Research Center (M.J.W., G.E.-B., K.L.K., E.T.), Madison, Wisconsin 53715; Biological Sciences, University of Wisconsin (M.J.W., E.N., A.A.), Whitewater, Wisconsin 53190; Department of Medicine, Tulane University (C.Y.B.), New Orleans, Louisiana 70112; and Department of Pediatrics, University of Wisconsin (E.T.), Madison, Wisconsin 53792

Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu.

Abstract

A decline in somatic function with aging in women is associated with a decrease in GH release and a loss of estrogen after menopause. As an initial step to establish a monkey model for the neuroendocrine mechanisms underlying somatopause and menopause, we have conducted three experiments in unrestrained aged (~25.7-yr-old) and young (~5.4-yr-old) female rhesus monkeys. GH release was pulsatile, and mean GH release and pulse amplitude were significantly lower in aged monkeys than in young monkeys. Injection of GHRH alone, GH-releasing peptide-2 alone, or the combination of both induced an increase in GH release in both age groups. The mean LH level, pulse amplitude, and baseline LH levels were significantly higher in aged animals than in young animals. Both estrogen and IGF-I levels were lower in aged than young monkeys. These results suggest that in female rhesus monkeys 1) there is a clear decline in circulating GH and IGF-I levels with aging; 2) GHRH and GH-releasing peptide-2 stimulate GH release synergistically; and 3) circulating LH levels increase as estrogen decreases with aging. These results indicate that the rhesus monkey is an excellent model for studies of the neuroendocrine mechanisms of aging.

A DECLINE IN SOMATIC function in association with female aging in higher primates is characterized by somatopause (1) and menopause (2). While menopause is due to cessation of cyclic ovulation, resulting in a decrease in circulating estrogen and an elevation of LH release, somatopause is due to decreases in GH and IGF-I with aging (3, 4). Both LH and GH are released in a pulsatile manner from the gonadotrophs and somatotrophs in the pituitary, respectively. LH release is stimulated by the hypothalamic decapeptide, LH-releasing hormone (LHRH), whereas GH release is regulated primarily by two hypothalamic peptides: GHRH and somatostatin (5, 6, 7). To date, however, aging-related changes in the mechanisms controlling GH release remain to be clarified.

It has been suggested that aging-related decreases in GH tone are the result of a decrease in hypothalamic stimulation by GHRH (8, 9, 10, 11) and a corresponding increase in hypothalamic inhibition by somatostatin (12, 13, 14, 15), although the relative importance of these two hypothalamic regulators is not fully understood. In addition, aging greatly alters the GH response to a class of synthetic peptides called GH-releasing peptides (GHRPs) that stimulate the somatotrophs and/or GHRH neurons to release GH with higher efficacy than the native stimulatory peptide GHRH (16, 17). Similarly, aging-related changes in LH and LHRH release have not been well studied. The loss of ovulatory cycles with aging and a subsequent decrease in circulating estrogen result not only in an increase in LH release (and presumably LHRH release), but in a reduction in spontaneous GH release and in the efficacy of GHRP stimulation of GH release (18). The degree to which estrogen contributes to GH release directly or indirectly through the regulatory peptides, GHRH and somatostatin, is unclear.

Despite a substantial number of clinical reports in humans, progress in understanding neuroendocrine mechanisms of hypothalamic aging has been hampered by the absence of a proper research model. The rhesus monkey is a potential model for human somatopause and menopause, because characteristics of the aging process are very similar. In addition, the rhesus monkey offers the ability to sample from the hypothalamus directly for the release of GHRH and somatostatin, which is not practical in human clinical research. Therefore, the present study was designed to establish a monkey model for neuroendocrine mechanisms in human aging. We characterized pulsatile GH release and LH release in aged female rhesus monkeys compared with young adult females, and we conducted a study evaluating the GH responsiveness to GHRP-2, GHRH, and a combination of these two peptides.

Materials and Methods

Animals

Nineteen healthy adult female rhesus monkeys (Macaca mulatta) of two age groups (young group: n = 9; average age, 5.4 yr; aged: n = 10; average age, 25.7 yr) were used in this study. Six (two aged and four young) of the monkeys were used in all three experiments, the remaining animals were used in one (experiment 2) or two experiments (experiments 1 and 3). No monkeys were killed during this project. Menstrual cycles were monitored by noninvasive daily sex skin observations, and all young monkeys exhibited regular menstrual cycles. Because some aged monkeys in our colony still maintain regular endocrine cycles, we chose only aged monkeys exhibiting acyclic (n = 4) or irregular (n = 6) menstrual cycles with a prolonged follicular phase. When cycling, monkeys were used only during the early follicular phase (within 7 d after the initiation of menstruation). All monkeys were adapted to the jacket-tether apparatus for several weeks before the surgery. This training included three or more separate sessions for each animal. The animals were housed in pairs, under controlled lighting (12 h of light, 12 h of darkness, lights on at 0600 h) and temperature (22 C), as described previously (19), and were provided a standard diet of Purina monkey chow (Ralston Purina Co., St. Louis, MO) at 0830 h daily, supplemented with fresh fruit several times per week. Fresh fruit feeding was eliminated on the day of the experiment. Water was available ad libitum. The protocol for this study was reviewed and approved by the animal care and use committee of University of Wisconsin, and all experiments were performed under the guidelines established by the NIH and USDA.

Experimental design

To obtain samples from a remote site without disturbing the research subjects, animals were implanted with indwelling catheters in their jugular vein (see below). The catheter was exteriorized through the tether-jacket system and was connected to a swivel device at the top of each cage through a wall to an adjacent room, i.e. the paired animals were kept in an isolated sound proof room with a one-way, see-through glass window from which we could observe their behaviors. Before the experiment, single blood samples (3.0 ml) from 2 successive d were obtained from all animals for LH, estrogen, and progesterone assays.

In experiment 1, to assess the age-related changes in circulating GH, 0.45-ml blood samples were collected at 10-min intervals for 10 h (1800–0200 h) from seven young (average age, 5.4 yr) and five aged (average age, 26.3 yr) female monkeys. Evening hours were chosen to examine GH release, because more pulses were reported in the dark phase, especially during slow wave sleep in primates (20, 21, 22, 23, 24, 25). After each blood sampling, plasma was separated by centrifugation, and the blood cells from the previous sample were resuspended in sterile saline and reinjected into the monkey. Plasma samples were stored at -80 C until assayed.

In experiment 2, to examine the pituitary responsiveness to human 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) (Bachem, King of Prussia, PA) and GHRP-2 (supplied by C. Y. Bowers), plasma samples were collected at 15-min intervals in six young (average age, 5.4 yr) and seven aged (average age, 26.7 yr) monkeys using methods similar to those described for experiment 1. After baseline sampling for 1.25 h, three challenges (GHRH, GHRP-2, and GHRH plus GHRP-2) were given through the catheter successively at 3-h intervals, and blood sampling was continued. The doses of GHRH and GHRP-2 were both 1 µg/kg body weight. The experiment was conducted from 0730–1730 h. To minimize the total period of sampling in aged monkeys, we did not examine the GH response to vehicle. Plasma was stored at -80 C until assayed by RIA for GH concentrations.

In experiment 3, to examine changes in the LH pulse pattern with aging, we also assessed LH levels in aliquots of the same samples collected for GH release in experiment 1. Before the experiment we collected serum samples twice on successive days from all animals for estrogen, progesterone, and IGF-I measurements.

Catheterization

Under isoflurane anesthesia, an indwelling catheter made from SILASTIC brand tubing (Dow Corning Corp., Midland, MI) was inserted into the jugular vein of the animal 2 d before the start of each experiment, as described previously (26, 27). The catheter was exteriorized through the back, and an extension of the catheter was passed through a cloth jacket with a tether-swivel system, allowing normal movement by the monkey. The catheter extension was connected to a three-way stopcock for blood sampling and injection. The patency of the catheters was maintained by constant infusion of 0.5 ml/h sterile saline containing 5 IU/ml heparin.

Hormone assays

The GH concentration in the plasma samples was measured in duplicate (50 µl in each) by RIA, using anti-GH serum (NIDDK hGH-2) at an incubation concentration of 1:440,000. The assay buffer was 0.1% gelatin in 0.01 M phosphate, 0.15 M NaCl, and 0.1% sodium azide, pH 7.40. The standard was NIDDK hGH-RP-1, and the trace was made by iodination of monkey GH. This RIA was a standard double antibody assay. The sensitivity of the GH assay was 0.1 ng/tube at 93% binding. The intraassay coefficient of variation was 10.6%.

The LH concentration in the plasma samples from experiment 1 was measured singly (100 µl) using an RIA with anti-LH serum (NICHHD R13, pool D; provided by Dr. G. Niswender), as described previously (26). The sensitivity of the LH assay was 2.0 ng/tube at 90% binding. The intraassay coefficient of variation was 4.9%.

Serum estradiol and progesterone concentrations were determined using RIA systems developed at the Wisconsin Regional Primate Research Center (28, 29).

Serum IGF-I levels were assessed using an RIA kit for human IGF-I (no. 40-2100, Nichols Institute Diagnostics, San Juan Capistrano, CA) after extraction with acid-ethanol precipitation.

Pulse detection

GH and LH pulses from experiments 1 and 3, respectively, were detected using the PULSAR algorithm (30). Pulses of GH and LH were determined using parameters similar to those previously reported (31), with cut-off criteria for G1, G2, G3, G4, and G5 of 3.8, 2.6, 1.9, 1.5, and 1.2 SD, respectively. The interassay standard variation for GH assay was described by the equation y = [0.14x2 - 0.49x + 11.57]/100. The interassay standard variation for LH assay was y = [2.77x + 16.69]/100, as described previously (27).

Statistical analysis

In experiments 1 and 3, mean GH and LH levels were calculated from the average of GH and LH values in individual animals. Age-related changes in mean GH and GH pulse parameters (interpulse interval, amplitude, and baseline levels) were examined by t test. Similarly, the effects of aging on LH levels and LH pulse parameters were examined by t test. In experiment 2, the GH responses to GHRH, GHRP-2, and the combination of both peptides were calculated as the difference between the peak GH response and the baseline GH levels during the 30-min period before treatment. The GH responses were then compared using two-way ANOVA with repeated measures, followed by a Student- Newman-Keuls post hoc analysis of log-transformed data. Statistical significance was attained at P < 0.05.

Results

Changes in GH secretion with aging

The release of GH in both aged and young female rhesus monkeys was pulsatile, as shown in examples from the two age groups (Fig. 1Go). GH pulses in aged monkeys (Fig. 1Go, A and B) were generally smaller than those in young monkeys (Fig. 1Go, C and D), although in aged monkeys an occasional high amplitude pulse was observed (Fig. 1Go, A and B). Analysis of the group data indicated that mean GH levels (3.41 ± 0.59 ng/ml) in aged monkeys were significantly (P < 0.05) lower than those (5.0 ± 0.5 ng/ml) in young adults (Fig. 2AGo). Similarly, the pulse amplitude of aged monkeys (2.8 ± 0.6 ng/ml) was significantly (P < 0.05) smaller than that (8.0 ± 2.1 ng/ml) in young monkeys (Fig. 2BGo). The interpulse interval (71.9 ± 9.5 min) in aged monkeys did not differ from that (66.8 ± 10.1 min) in young monkeys (Fig. 2CGo), and there was no difference in the baseline GH levels (2.5 ± 0.2 ng/ml in aged; 2.5 ± 0.3 ng/ml in young; Fig. 2DGo).



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  		Figure 1. GH release profiles in two aged (A and B) and two young (C and D) female rhesus monkeys. Representative cases from each group are shown. Arrows indicate pulses identified by PULSAR algorithm.

 


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  		Figure 2. Changes in mean GH levels (A), GH pulse amplitude (B), GH interpulse interval (C), and baseline GH release (D) associated with aging. Aged female group, n = 5; young group, n = 7. *, P < 0.05, aged vs. young.

 
Effects of GHRH, GHRP-2, and their combination

GHRH (1 µg/kg) in both aged (Fig. 3Go) and young (Fig. 4Go) monkeys induced a small GH increase from the baseline level, but it was consistent among individuals. The mean GH level after GHRH was significantly higher than that before GHRH in aged (P < 0.05), but not in young, monkeys (Fig. 5Go). There was no significant difference in the GH response between the two age groups (Fig. 5Go). The GH response to GHRP-2 (1 µg/kg) was also small in both young and aged animals (Figs. 3Go and 4Go), although there was more individual variation among the aged animals (Fig. 3Go). The effects of GHRP-2 on mean GH release in both groups were significant (P < 0.05 for aged; P < 0.01 for young; Fig. 5Go). However, there was no difference in the GH response to GHRP-2 between the two age groups (Fig. 5Go). In contrast, the GH response to the combination treatment of GHRH and GHRP was substantially larger than that to GHRH or GHRP-2 alone in both young and aged monkeys (Figs. 3Go and 4Go), and this effect was significant (P < 0.01 for aged and P < 0.02 for young). Moreover, the response to the combination treatment of GHRH and GHRP was significantly larger than that to GHRH alone in both aged and young females (P < 0.05 for both). The GH response to the combination treatment in young females was also significantly larger than that to GHRP-2 alone (P < 0.05 for both). Again, there was no significant difference in the combination treatment when the two age groups were compared.



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  		Figure 3. Effects of GHRH, GHRP-2, and GHRH plus GHRP-2 on GH release in two aged female monkeys.

 


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  		Figure 4. Effects of GHRH, GHRP-2, and GHRH plus GHRP-2 on GH release in two young females monkeys.

 


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  		Figure 5. Comparison of GH responses to GHRH, GHRP-2, and GHRH plus GHRP-2 challenges in aged and young monkeys. Aged female group, n = 7; young female group, n = 6. *, P < 0.05 vs. before challenge; **, P < 0.02 vs. before challenge; ***, P < 0.01 vs. before challenge; a, P < 0.05, GHRH plus GHRP-2 vs. GHRH alone; b, P < 0.05, GHRH plus GHRP-2 vs. GHRP-2 alone. None of the parameters was different between the two age groups.

 
Changes in LH secretion with aging

The release of LH in both aged and young female rhesus monkeys was pulsatile, as shown by examples from the two age groups (Fig. 6Go). In general, LH pulses in aged monkeys (Fig. 6Go, A and B) were larger than those in young monkeys (Fig. 6Go, C and D). Analysis of the group data indicated that the mean LH level (37.4 ± 10.0 ng/ml) in aged monkeys was significantly (P < 0.02) higher than that (7.2 ± 3.1 ng/ml) in young adults (Fig. 7AGo). Similarly, the pulse amplitude in aged monkeys (41.5 ± 9.2 ng/ml) was significantly (P < 0.01) larger than that (3.7 ± 1.5 ng/ml) in young monkeys (Fig. 7BGo), and the baseline LH level (25.6 ± 9.9 ng/ml) in aged monkeys was also higher than that (5.7 ± 2.6 ng/ml) in young monkeys (Fig. 7DGo). The interpulse interval (55.6 ± 4.8 min) in aged monkeys did not differ from that (71.7 ± 5.7 min) in young monkeys (Fig. 7CGo).



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  		Figure 6. LH release profiles in two aged (A and B) and two young (C and D) female rhesus monkeys. Representative cases from each group are shown. Arrows indicate pulses identified by PULSAR algorithm.

 


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  		Figure 7. Effects of aging on mean LH levels (A), LH pulse amplitude (B), LH interpulse interval (C), and baseline LH release (D). Aging group, n = 5; young group, n = 7. *, P < 0.05; **, P < 0.02; ***, P < 0.01 (aged vs. young).

 
Changes in estradiol, progesterone, and IGF-I levels (Table 1Go)

Before the three experiments, we collected serum samples from all animals and measured LH, estradiol, progesterone, and IGF-I. The mean LH level (43.3 ± 11.8 ng/ml) in the aged monkeys (n = 9) was significantly higher (P < 0.025) than that (15.1 ± 1.6 ng/ml) in the young monkeys (n = 10; Table 1Go). Estradiol levels in aged monkeys were also significantly lower (P < 0.05) than those in young monkeys (n = 9). Progesterone levels were not different between the two groups. The mean IGF-I level in aged monkeys (134 ± 23 ng/ml) was significantly (P < 0.01) lower than that (281 ± 32 ng/ml) in young monkeys (Table 1Go).


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  		Table 1. Changes in gonadal steroids with aging

 
Discussion

Female rhesus monkeys at 25–30 yr of age are estimated to be equivalent to human females at 65–80 yr of age. This estimate is based on a number of factors: 1) female rhesus monkeys have a longer reproductive life than humans; 2) the maximum life expectancy of the rhesus monkey (M. mulatta) in captivity is approximately 40–45 yr (32); and 3) menopause in our colony females occurs at about 26–28 yr of age (32), although reproductive aging in the macaque family appears to start as early as 18 yr of age, as shown in Macaca fuscata (Japanese macaque), based on extensive hormone profiles and fecundity records (33).

The present study represents an important step in establishing normal release profiles for GH and LH in aging female rhesus monkeys. The rhesus monkey remains an extremely important experimental model for many investigations in neuroendocrinology. The results in the present study establish the foundation for future studies in which aging changes in the hypothalamic regulatory peptides, GHRH, somatostatin, ghrelin, and LHRH, in nonhuman primates can be studied as a model. The rhesus monkey model for studying mechanisms of human aging is very important, because characteristics of the aging process are very similar in rhesus and human, and the rhesus monkey offers the ability to sample from the hypothalamus directly for the release of GHRH and somatostatin, which is not available from human clinical observation.

In the first experiment we confirmed that in female rhesus monkeys, as reported in humans, GH release is pulsatile and decreases with aging. In both men and women an aging-related decrease in mean GH or integrated GH levels has been reported (20, 24, 25, 34, 35, 36). A similar finding in male monkeys has also been reported (23). To our knowledge, however, this is the first observation of pulsatile GH release profiles and aging-related decline in GH in female nonhuman primates. In comparisons between pulsatile GH release profiles from young and aged monkeys, mean GH levels and GH pulse amplitude were lower in aged monkeys than in young monkeys, but baseline GH levels and the GH interpulse intervals were not different. In some human studies only the pulse amplitude component decreased with aging (15, 35, 37), whereas in another study only the frequency component decreased (36) with aging. In male rhesus monkeys the amplitude of GH pulses decreased with aging during both light and dark phases, whereas the frequency decreased with aging during the dark phase (23). In the present study in female monkeys we obtained samples only during the dark phase, but we did not observe changes in pulse frequency with aging. It is possible that a longer period of blood sampling (24 h) may elucidate changes in pulse frequency, as in the present study samples were obtained for approximately 10 h.

In the second experiment we examined the effects of GHRH, GHRP-2, or a combination of both on GH release in aged and young monkeys. Although GHRH alone and GHRP-2 alone induced modest, but consistent, GH responses, the combination of both GHRP-2 and GHRH resulted in much larger GH responses in both age groups. A similar synergistic effect of GHRH and GHRP-2 has been reported in humans (38, 39, 40). Moreover, there are numerous reports in humans indicating that GH responses to either GHRH or GHRP-2 alone or a combination of both decrease with aging (41, 42, 43, 44, 45, 46, 47, 48). In our study neither the effects of aging on GH responses to GHRH and GHRP-2 alone nor the effects of aging on the synergistic actions of GHRH and GHRP-2 were observed. Although for this difference a species difference in monkeys and humans cannot be excluded, it is possible that the small number of animals used in this study may be a significant problem. In fact, there was a trend for the GH response to the combination of GHRH and GHRP-2 in aged animals to be smaller than that in young animals in our study, but the responses were not significant because of a large individual variation. It is also possible that small responses to GHRH and GHRP-2 alone may be due to an elevated level of IGF-I, because these challenges were given around or shortly after feeding.

Over the past 15 yr a considerable amount of work has been performed evaluating a class of GH-stimulating molecules, the GHRPs, primarily in human subjects. GHRPs are thought to stimulate both the hypothalamus and pituitary (49). GHRPs administered either peripherally or centrally through intracerebroventricular cannula stimulated c-Fos activity in neurons of the arcuate nucleus (50). GHRPs bind to hypothalamic tissue and stimulate GH-releasing activity (51, 52). GHRPs have also been shown to bind to pituitary tissue (51, 52) and stimulate GH release from somatotropes in culture (16). Receptors for GHRP have been cloned (53), and ghrelin, an endogenous ligand for GHRP receptors, has been isolated (54).

In the third experiment we examined the changes in pulsatile LH release with aging. Mean LH in aged females was significantly higher than that in young females. The pulse amplitude and baseline LH levels were significantly higher in aged animals, but the pulse frequency did not differ. The elevated LH levels in aged females were attributed to the decreased estrogen levels. These observations are consistent with those reported in humans (55).

It is plausible that the aging-related decrease in GH release is due to a decrease in circulating estrogen after menopause. For example, studies in young women indicate that there is a positive correlation between estradiol and mean GH levels during the menstrual cycle (56, 57, 58). Estradiol and mean GH levels are positively correlated among a large group of subjects, including men and women at various ages (35, 56). The mean concentration and the pulse amplitude of GH release are higher in women than in men (59, 60). Long-term treatment with estrogen in patients with hypogonadotropic hypogonadism increases circulating GH levels (61). In addition, the ability of GHRH to stimulate GH release is proportional to circulating estrogen levels (15); ovariectomy decreases the effect of GHRH on GH release, and estrogen treatment restores it (62); and the GH response to GHRH in women is also higher than that in men (63). Estrogen also increases GH responses to GHRPs and other secretagogues (15, 57). These clinical data indicate that estrogen modulates the release of GHRH and somatostatin from the hypothalamus and GH release from somatotrophs. However, a study treating postmenopausal women and premenopausal women due to premature ovarian failure with estrogen replacement therapy indicate that a reduced GH release and a reduced GH response to GHRH or secretagogues with aging are not simply explained by a decrease in estrogen after menopause (64). GH levels with estrogen supplementation in older postmenopausal women were different from those in younger postmenopausal women (46, 65, 66), and transdermal administration to postmenopausal women did not sufficiently increase GH levels compared with the effect of estrogen administration with oral administration (67). Therefore, it is hypothesized that the GH reduction in aging is not solely due to estrogen reduction after menopause, but aging of the hypothalamo-pituitary axis that controls GH release is also involved.

The hypothalamic mechanism of the aging-related decrease in GH release remains speculative. It has been suggested that aging of the somatotrophs is partly responsible (41, 42, 43, 44, 60, 68), and aging of the regulatory mechanism of GH release is also involved. However, in the latter it is still unclear whether 1) the release of GHRH, a stimulatory peptide, decreases with aging (24, 35, 37, 69, 70); 2) the release of somatostatin, an inhibitory peptide, increases with aging (48, 71); or 3) combination of the two (72, 73). Although numerous studies support each possibility, we need direct measurements of GHRH and somatostatin in young and aged primates. A preliminary study from our laboratory indicates that the aging-related decrease in GH release appears to be due to both a decrease in GHRH and an increase in somatostatin (74). Nonetheless, because circulating estrogen affects GH release through multiple sites of action, as discussed above, the question remains as to how much the aging-related reduction in GH release in female primates is attributed to changes in hypothalamic aging and to the aging-related reduction in estrogen.

Acknowledgments

We thank the animal care staff, particularly Harry Pape, for the professional care of our research animals. Denny Mohr provided expert assistance during surgical procedures.

Footnotes

This work was supported by NIH Grants AG-14972 and RR-00167 (to E.T.). This study is Publication 42-003 from the Wisconsin Regional Primate Research Center.

Abbreviations: GHRP-2, GH-releasing peptide-2; LHRH, LH- releasing hormone.

Received April 30, 2002.

Accepted August 14, 2002.

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