Aging-Related Changes in in Vivo Release of Growth Hormone ...

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The Journal of Clinical Endocrinology & Metabolism 88(2):827– 833 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-021568

Aging-Related Changes in in Vivo Release of Growth Hormone-Releasing Hormone and Somatostatin from the Stalk-Median Eminence in Female Rhesus Monkeys (Macaca mulatta) SHINICHIRO NAKAMURA, MASAHARU MIZUNO, HIDEKI KATAKAMI, ANDREA C. GORE, EI TERASAWA

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Wisconsin National Primate Research Center (S.N., M.M., E.T.) and Department of Pediatrics (E.T.), University of Wisconsin–Madison, Madison, Wisconsin 53715; Department of Neurobiology and Geriatrics (A.C.G.), Mount Sinai School of Medicine, New York, New York 10029; and Department of Internal Medicine (H.K.), Miyazaki Medical College, Miyazaki 889-1692, Japan GH release decreases with aging in primates. However, it is unclear whether the age-related decrease in GH release is due to a decrease in stimulatory GHRH or an increase in inhibitory somatostatin (SS) from the hypothalamus. In the present study, we measured the release of GHRH and SS in the stalkmedian eminence of conscious aged (n ⴝ 7, 27.0 ⴞ 0.7 yr old) and young adult female monkeys (n ⴝ 12, 5.0 ⴞ 0.3 yr old) using the push-pull perfusion method. Mean GHRH levels during morning (0600 –1200 h) and evening (1800 –2400 h) in aged monkeys were 3- to 4-fold lower than in young monkeys. Pulse analysis indicated that pulse frequency, pulse amplitude, and

baseline GHRH release in aged monkeys were much lower than in young adults. In contrast, mean SS levels in aged monkeys during mornings and evenings were 2-fold higher than in young monkeys. Pulse analyses indicated that amplitude and baseline levels of SS were significantly higher in aged monkeys than in young adults. There were no significant changes in the pulse frequency of SS release. Therefore, the aging-related decrease in GH release is due to a substantial decrease in GHRH release and an increase in SS release from the hypothalamus. (J Clin Endocrinol Metab 88: 827– 833, 2003)

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UMEROUS STUDIES IN humans indicate that GH release decreases with aging (1–3). Similarly, circulating GH levels in rhesus monkeys as well as in rodents decrease with aging (4 –7, 9, 10). This GH decrease is regarded as a cause of the somatic decline, such as a reduction of protein synthesis, a decrease in lean body mass and bone mass, and decline of the immune functions (1, 11–13). It has been generally accepted that GH release is regulated primarily by two hypothalamic peptides, GHRH and somatostatin (SS): GHRH stimulates GH release and SS inhibits GH release (14 –18). However, to date it is unclear what causes the aging-related GH decrease. The aging changes could occur at the pituitary or hypothalamic level. The number and size of GH-producing cells in the human pituitary decrease with aging (19), and the GH response to GHRH indicates that the pituitary somatotropes become less sensitive to GHRH in aged humans and rats (20 –25). A line of reports indicates that the hypothalamus undergoes age-related changes. In fact, the question of whether the decrease in GH with aging is due to a reduction in GHRH, an increase in SS, or a combination of both has been debated for more than a decade. For example, it has been suggested that the aging-related decrease in GH release is due to a decline of GHRH release because studies in rats indicate that both GHRH peptide and mRNA expression

decrease with aging, whereas SS mRNA expression exhibits little change (26 –28). In humans as well, impairment of the GHRH-releasing system with aging has also been suggested because there is a decrease in only the amplitude component, but not the frequency component, of GH pulses (29, 30), and less recovery of GH release from continuous infusion of SS inhibition is observed in elderly subjects than in young adults (31). Alternatively, it has been suggested that the reduction in GH release with aging is due to the increased tone of SS release since the reduced GH response to GHRH in aged men is also restored by administration of l-arginine (22), which is thought to suppress SS release (32) because administration of antiserum to SS resulted in GH release in aged rats equal to or greater than that seen in young rats (33). More recently it has been hypothesized that both a decrease in GHRH and an increase in SS are responsible for the aging-related GH decrease (34 –36). One of the most significant problems in understanding the control mechanism of GH release in primates is the paucity of methods for assessing the release of GHRH and SS directly. Therefore, the goal of this study was to measure the aging-related changes in the release of GHRH and SS in the stalk-median eminence (S-ME), where their neuroterminals contact the portal vessels (37). We accomplished this goal using a push-pull perfusion method, based on our extensive experience in measuring LHRH of conscious female rhesus monkeys (38, 39).

Abbreviations: PPP, Push-pull perfusion; S-ME, stalk-median eminence; SS, somatostatin.

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Materials and Methods Animals Two groups of female rhesus monkeys (Macaca mulatta) were used in the present study: Aged monkeys (n ⫽ 7) at age of 27.0 ⫾ 0.7 yr and young adults (n ⫽ 12) at 5.0 ⫾ 0.3 yr. Although all young females exhibited regular menstrual cycles, aged females were acyclic (n ⫽ 4) or exhibited irregular menstrual cycles (n ⫽ 3). If animals were cyclic, perfusate samples were obtained during the early follicular phase (within 7 d after the observation of menstruation). Aged monkeys were born in the wild or laboratories within the United States but housed in Wisconsin National Primate Research Center (WNPRC), University of Wisconsin–Madison. All young adult monkeys were born and housed at WNPRC. All females were kept in a cage (172 ⫻ 86 ⫻ 86 cm) in pairs, and the room was kept at 22 C with 12 h of light (0600 –1800 h) and 12 h of darkness (1800 – 0600 h). Purina monkey chow was fed daily, supplemented with fruits three times a week. Water was available ad libitum. The protocol for this study was reviewed and approved by the Animal Care and Use Committee, University of Wisconsin, and all experiments were conducted under the guidelines established by the NIH and USDA.

Push-pull perfusion Before the push-pull perfusion (PPP) experiments, the monkeys were implanted with cranial pedestals under isoflurane anesthesia (38, 39). The animals were allowed to recover for at least 4 wk before the start of PPP experiments, during which time they were gradually adapted to the primate chair and experimental situation. The method for PPP of the S-ME in conscious monkeys was similar to that described previously (38). Briefly, the concentric push-pull cannula was stereotaxically inserted with the aid of a radioventriculogram, using a hydraulic microdrive unit (MO95-B, Narishige, Tokyo, Japan), into the S-ME and the area confined to 1.0 mm posterior and ventral to the tip of the infundibular recess and laterally within 1.0 mm of the midline. On the third day after cannula insertion, a modified Krebs-Ringer phosphate buffer solution was infused to the S-ME through the push (inner) cannula at a rate of 23 ␮l/min with a peristaltic pump (Gilson, Middleton, WI). Perfusate was collected in 10-min fractions on ice from the pull (outer) cannula using an identically calibrated pump. This perfusion was carried out for 6 h in the morning, from 0600 h to 1200 h (lights on), and 6 h in the evening, from 1800 h to 2400 h (lights off). The order of the morning and evening sampling sessions was randomized. Each collected sample was divided into a 150-␮l vial for GHRH assay and a 50-␮l vial for SS assay. All samples were stored at ⫺70 C until RIA.

Nakamura et al. • Age Changes in in Vivo Release of GHRH and Somatostatin

groups during the morning and evening was statistically compared using two-way ANOVA. Similarly, the difference of SS release in each parameter between two age groups during the morning and evening was compared using two-way ANOVA. Analyses showing a P value of more than 0.05, but less than 0.1, were transformed to log (x⫹1) because of very high individual variations. Specifically, GHRH amplitude during evening and SS baseline levels during morning were transformed. The transformed value was further analyzed by one-way and two-way repeated ANOVA. The number of reciprocal GHRH peaks against SS troughs or GHRH peaks coinciding with SS peaks were counted from overlay graphs of GHRH and SS release in each animal; the percent of reciprocal peaks or coincidental peaks from the total peak numbers in each age group were calculated. Statistical significance was calculated using Mann-Whitney U test. All statistical significance was attained at P less than 0.05.

Results GHRH release in aged and young adult monkeys

GHRH release in aged female rhesus monkeys was pulsatile with low amplitudes and low baselines (Fig. 1). Similarly, GHRH release in young female rhesus monkeys was pulsatile, but its pulse amplitude and baseline levels were higher than those in aged monkeys (Fig. 2). There was no apparent variation in the GHRH release pattern because of the time of the day for either age group (Figs. 1 and 2). Statistical analysis indicates that mean GHRH during morning and evening in aged monkeys was approximately 4-fold lower than that in young monkeys (P ⬍ 0.005, Fig. 3A). There was no difference in the morning and evening levels in either age group. Pulse analysis further suggests that baseline levels (Fig. 3B), pulse amplitudes (Fig. 3C), and pulse frequencies (Fig. 3D) in aged monkeys were all significantly

RIA Collected aliquots were used for RIAs. GHRH was measured using human GHRH assay kit (Peninsula Laboratories, Inc., Belmont, CA). SS was measured with rabbit antiserum against SS (40) as a primary antibody and synthetic SS (Peninsula Laboratories, Inc.) as a radiolabeled antigen. Synthetic SS was also used for a standard curve. The antigenantibody complex was precipitated with a goat antiserum against rabbit IgG. Sensitivities of these assays were 2.6 pg/tube for GHRH and 0.25 pg/tube for SS, and intra- and interassay coefficients of variation were 8.46% and 15.63% for GHRH, and 8.35% and 11.29% for SS, respectively.

Data analyses Both GHRH and SS were measured in the aliquots of all seven aged monkeys and all 12 young monkeys except for one monkey in which only SS was measured. GHRH and SS pulses in perfusates were identified by the PULSAR program (41), as described previously. The cut-off criteria for pulse determination, G1, G2, G3, G4, and G5 were 4.4, 2.6, 1.92, 1.46, and 1.13, respectively. Parameters of pulsatile GHRH and SS release were calculated for each sampling session as follows: 1) mean GHRH and SS release: the mean of all GHRH and SS values during the 6-h sampling; 2) baseline level: the mean of all trough values from GHRH and SS, i.e. the lowest value between peaks; 3) pulse amplitude: the mean of all amplitudes derived from the difference between peak and trough; and 4) pulse frequency: the mean number of pulses per hour. The difference of GHRH release in each parameter between two age

FIG. 1. Representative examples of pulsatile GHRH release from two aged female monkeys (A and B), with the same scale as young females shown in Fig. 2. The case (B) is shown with an enlarged scale in C to demonstrate GHRH pulses. The arrows indicate GHRH pulses identified by the Pulsar algorithm.


FIG. 2. Representative examples of GHRH release from two young adult female monkeys (A and B), with the same scale as aged females shown in Fig. 1. The case (B) is shown with a full scale to demonstrate the whole picture in C. The arrows indicate GHRH pulses identified by the Pulsar algorithm.

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FIG. 3. Aging-related change in GHRH release during 6 h in the morning (open bars) and 6 h in the evening (dotted bars). Mean (A), baseline (B), pulse amplitude (C), and pulse frequency (D) are shown. Note that mean, baseline, pulse amplitude, and pulse frequency of GHRH release in aged monkeys (n ⫽ 7) were all significantly lower than those in young adult monkeys (n ⫽ 11). ***, P ⬍ 0.005; **, P ⬍ 0.01; *, P ⬍ 0.05, vs. young adult monkeys at corresponding morning or evening data.

lower than those in young monkeys (baseline levels: P ⬍ 0.01 for morning, P ⬍ 0.05 for evening; pulse amplitude: P ⬍ 0.01 for both morning and evening; pulse frequency: P ⬍ 0.05 for morning and P ⬍ 0.005 for evening). There were no significant differences in evening and morning values for all pulse parameters examined. SS release in aged and young adult monkeys

SS release in aged rhesus monkeys was pulsatile with high baseline levels and large pulse amplitudes (Fig. 4). SS release in young females was also pulsatile, but SS pulse amplitude in young females was much lower than that in aged females (Fig. 5). There was no apparent difference in the morning and evening patterns. Statistical analysis indicated that mean SS in aged monkeys during morning and evening was approximately 2-fold higher than young monkeys (for both P ⬍ 0.05, Fig. 6A). Again, there was no difference in the morning and evening levels in either age group. Pulse analysis further suggested that both baseline levels (Fig. 6B) and pulse amplitudes (Fig. 6C) in aged monkeys were significantly larger than those in young monkeys (baseline levels: P ⬍ 0.01 for morning, P ⬍ 0.05 for evening; pulse amplitude: P ⬍ 0.01 for morning, P ⬍ 0.05 for evening). The pulse frequency in aged and young females in both morning and evening was not significantly different (Fig. 6D). There were no significant differences in morning and evening values of any pulse parameter examined.

FIG. 4. Representative examples of pulsatile SS release from two aged female monkeys (A and B). The arrows indicate SS pulses identified by the Pulsar algorithm.

Because the examples for SS release shown in Figs. 4 and 5 were assayed in aliquots of the same samples used for GHRH assay (Figs. 1 and 2, respectively), we assessed the possible reciprocal relationship. Although in aged monkeys 18 –20% of GHRH peaks were reciprocal to SS troughs, in young monkeys 34 –50% of GHRH peaks were reciprocal to

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in young females, although the difference did not attain statistical significance (Table 1). There was no consistent correlation between mean values of GHRH levels and SS levels in individual cases (data not shown). Comparison of sampling location in the S-ME

Cannula tips of PPP in all monkeys were located within the area confined 1 mm2 below the base of the third ventricle, in which the S-ME is located, as seen in Fig. 7. We also noted that sampling location in aged and young monkeys overlapped considerably. Thus, the sampling location does not appear to be a factor for the individual differences observed for GHRH release in young monkeys and SS release in aged monkeys and also age-related differences in GHRH and SS levels. Discussion FIG. 5. Representative examples of SS release from two young adult female monkeys (A and B). The arrows indicate SS pulses identified by the Pulsar algorithm.

FIG. 6. Aging-related change in SS release during 6 h in the morning (open bars) and 6 h in the evening (dotted bars). Mean (A), baseline (B), pulse amplitude (C), and pulse frequency (D) are shown. Note that mean, baseline, and pulse amplitude of SS release in aged monkeys (n ⫽ 7) were significantly higher than those in young adult monkeys (n ⫽ 12), whereas pulse frequency of SS release did not differ between two age groups. ***, P ⬍ 0.005; *, P ⬍ 0.05 vs. young adult monkeys at corresponding morning or evening data.

SS troughs (Table 1). The rate of reciprocal GHRH peaks in morning aged females was significantly (P ⬍ 0.05) less that in morning young females. Moreover, although a few GHRH peaks lagged with SS troughs by 10 min, there were essentially no GHRH peaks that proceeded SS troughs by 10 min. Interestingly, a higher rate of GHRH peaks in aged females tended to be coincidental with SS peaks, compared with that

In the present study, we found that GHRH release in aged monkeys was 3- to 4-fold lower than in young adults females, whereas SS release in aged monkeys was 2-fold higher than in young adults. To our knowledge, this is not only the first study measuring GHRH and SS levels from the S-ME of female rhesus monkeys but also the first report assessing the aging-related changes in release of the hypothalamic hormone GHRH and SS in any species. The results of the present study correspond well with our previous findings (5) that GH release in aged female monkeys was lower than in young adult females. Indeed, our findings are interpreted to mean that the aging-related decrease in GH release is due to a substantial decrease in stimulatory GHRH release and an increase in inhibitory SS release from the hypothalamus. A hypothesis has been proposed that GH release is under the tonic inhibition of SS and that SS and GHRH are released in reciprocal 3- to 4-h cycles from the median eminence into the portal circulation (16, 17). This is based on results indicating that, in rats, the response to GHRH is larger during the GH peak than the GH trough and infusion of SS antibody increases the GHRH-induced GH release with an equally large response during both the GH peak and trough (16). This hypothesis is supported by an experiment with direct measurements of GHRH and SS in anesthetized rats: A slight suppression of SS was accompanied by an increased release of GHRH, and immunoneutralization of SS resulted in an increase in GHRH release (18). In human studies as well, GH release appears to be periodically suppressed by SS. GH pulses were clearly observed in a patient with an ectopic GHRH-secreting tumor as well as in normal men given a continuous infusion of GHRH (42). Nevertheless, a study with direct measurement of GHRH and SS from portal blood collection, accompanied by simultaneous blood sampling for GH assessment in unanesthetized sheep, indicated that although GH pulses were associated with GHRH pulses, changes in GH pulses were not significantly associated with SS pulses under normal physiological conditions (43– 45). Because in the study by Thomas et al. (44), restricted feeding resulted in increased GH release accompanied by decreased SS release, without changing the GHRH pulse pattern, SS appears to suppress the GH response to GHRH at the level of the somatotropes; therefore, a tonic recip-



TABLE 1. The rate of GHRH peaks with reciprocal SS troughs or GHRH peaks with coincidental SS peaks Aged females (n ⫽ 7)

Total GHRH peak number No. of GHRH peaks that coincide with SS troughs No. of GHRH peaks that lag SS troughs by 10 min No. of GHRH peaks that proceed SS troughs by 10 min No. of GHRH peaks that coincide with SS peaks a b

Young adult females (n ⫽ 11)

Morning

Evening

Morning

Evening

17 3 17.7%a 2 11.8% 0 0% 10 58.8%b

15 3 20.0% 3 20.0% 0 0% 5 33.3%

60 30 50.0% 3 5.0% 1 1.7% 14 23.3%

70 24 34.3% 6 8.6% 1 1.4% 18 25.7%

P ⬍ 0.05 vs. morning young females. P ⬍ 0.06 vs. morning young females.

FIG. 7. Sampling locations in the S-ME from aged (open triangles) and young (closed circles) monkeys are shown. Note that Cannula tips of PPP in all monkeys were located within the area confined 1 mm2 below the base of the third ventricle, at which the S-ME is located.

rocal relation exists between SS release and GHRH release under certain conditions in sheep. In the present study, we did not find any reciprocal relationship between mean GHRH and SS levels in individual animals. However, overlay graphs of GHRH and SS release indicate that 34 – 50% of GHRH pulses in young monkeys had a one-to-one reciprocal pattern against SS pulses, whereas 18 –20% of GHRH pulses in aged monkeys had a one-to-one reciprocal pattern against SS pulses. Interestingly, a lesser reciprocal relationship between GHRH and SS pulses in aged females is due to not only low GHRH levels but also the fact that more GHRH peaks occur coinciding with SS peaks. Therefore, the issue of inhibitory and stimulatory controls of GH release appears to be more complex than what has been proposed by Tannenbaum et al. (16, 17).

The nocturnal increase in circulating GH has been reported in humans, rats, and rabbits (46, 47). It is important to note that GH release occurs in association with slow-wave sleep (48, 49). The nocturnal GH increase is assumed to be due to activity changes in hypothalamic GHRH and/or SS neurons. For example, a higher nocturnal mRNA expression in the hypothalamus along with the suppression of nocturnal GH increase under the infusion of an antibody to GHRH was observed in rats (50). In humans, the administration of an antagonist for GHRH receptors decreased evening GH levels, but not morning GH levels, with more prominent effects in aged men than young adult men (51, 52), indicating that GHRH release from the hypothalamus is a stimulatory peptide for the nocturnal GH increase. In male rhesus monkeys, frequency of GH release decreased with aging only during the dark phase, whereas the amplitude of GH pulses decreased with aging during both light and dark phases (4). In the present study, we did not find significant nocturnal changes in the release of GHRH and SS, although the mean release, baseline levels, amplitude, and pulse frequency of GHRH release in aged monkeys were all lower than those in young monkeys, and the mean release, baseline levels, and amplitude of SS release in aged monkeys were higher than those in young monkeys. The reason for the absence of difference in morning and evening levels of GHRH release (and SS release) in this study is unclear. If we missed detecting it in the present study, it is possible that the animals may not be fully asleep, even though the light of the experimental room was off during evening sampling. Alternatively, to detect the difference we may need a longer period of sampling for both morning and evening. Aging-related changes in GHRH and SS neurons have been reported in rats. With immunocytochemistry the number of GHRH neurons and intensity of immunoreactive GHRH neurons did not differ between aged and young rats, but the intensity of the GHRH-immunoreactive fibers in the median eminence of aged rats was significantly lower than those in young rats (26). Similarly, in a study with in situ hybridization, expression of GHRH transcription in rats also decreased with aging (28, 53). These reports support the concept that impairment of GHRH neurons with aging might be a cause of a substantial decrease in pulsatile GHRH release in aged female rhesus monkeys. Clearly, similar histological studies with aging in the rhesus monkey remain to be conducted. In contrast, there are contradicting reports on the

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aging-related changes in SS neurons. One study (26) reported that neither the number of SS neurons nor the intensity of SS immunoreactivity was changed between aged and young rats, whereas another study (27) indicated that SS mRNA expression decreased with aging. These reports do not agree with our observations on the aging-related increase in SS release, although changes in quantity of the transcription does not always reflect the changes in quantity of its product (peptide) and peptide release (54). Interestingly, in the present study, although the GHRH frequency was significantly reduced in aged monkeys, there was no aging-related change in the SS frequency among the two age groups. This may be indicative of more severe aging-related changes in the GHRH neurosecretory system, rather than in the SS neurosecretory system, in control of GH release, corresponding to more consistent aging changes in rat GHRH neurons than rat SS neurons. Nonetheless, neurochemical studies on individual SS neurons with aging in the rhesus monkey remain to be conducted. Evidence indicates that a major determinant of circulating GH levels and GH responsiveness to GHRH is estrogen: There is a positive correlation between estradiol and mean GH levels during the menstrual cycle in women (55), and estradiol and mean GH levels are also positively correlated among a large group of subjects, including men and women at various ages (29). Long-term treatment with estrogen in patients with hypogonadotropic hypogonadism increases circulating GH levels (56). Ovariectomy decreases the effect of GHRH on GH release, and estrogen treatment restores it (57). Estrogen also increases the GH response to GH-releasing peptides and other secretagogues (55, 58). In these human studies, estrogen action on the somatotrope site cannot be excluded. Nonetheless, it is plausible that a reduction in GHRH release and an increase in SS release in the S-ME in this study are due to a decrease in estrogen after menopause. In fact, in our previous study (5), estrogen levels in aged monkeys at similar ages of the present study were significantly lower than those in young adults. In the future we need to study the degree to which the estrogen contributes to aging-related neurohormone release and the degree to which aging itself causes changes in GHRH neurons and SS neurons. In summary, in the present study, we found that the agingrelated decrease in GH release is due to a substantial decrease in GHRH release and an increase in SS release from the hypothalamus. The PPP method employed in this study with nonhuman primates is a very useful tool for the investigation of the control mechanism of GH release. In particular, the investigation into how estrogen reduction influences the aging-related changes in the release of GH, GHRH, and SS, and how other endogenous and exogenous secretagogues, such as ghrelin, GH-releasing peptide-2, and l-arginine, affect the release of GHRH and SS will not be discovered without this approach. We believe that our current findings shed light on the current understanding of the control mechanism of GH release in aged primates. Acknowledgments We thank Bret W. Engnell, Kim L. Keen, Frederick H. Wegner, and Dennis Mohr for technical assistance; Harold M. Pape for animal care;


and Drs. Iris D. Bolton, Kevin G. Brunner, and Carol L. Emerson for clinical veterinary care. Received October 7, 2002. Accepted November 15, 2002. Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin National Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: [email protected]. This work (publication no. 42-009 from the Wisconsin Regional Primate Research Center) was supported by NIH Grants AG17942 and RR00167 (to E.T.) and AG16765 (to A.C.G.). Present address for S.N.: Department of Veterinary Pathology, Nippon Veterinary and Animal Science University, Musashino, Tokyo, Japan. Present address for M.M.: Department of Integrative Physiology, Kyushu University, Fukuoka, Japan.

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