Decrease in Luteinizing Hormone Pulse Frequency during a Five-Hour Peripheral Ghrelin Infusion in the Ovariectomized Rhesus Monkey

The Journal of Clinical Endocrinology & Metabolism, Nov 2004

Ghrelin, a nutrition-related peptide secreted by the stomach, is elevated during prolonged food deprivation. Because undernutrition is often associated with a suppressed reproductive axis, we have postulated that increasing peripheral ghrelin levels will decrease the activity of the GnRH pulse generator. Adult ovariectomized rhesus monkeys (n = 6) were subjected to a 5-h iv human ghrelin (100- to 150-μg bolus followed by 100–150 μg/h) or saline infusion, preceded by a 3-h saline infusion to establish baseline pulsatile LH release. Blood samples were collected at 15-min intervals throughout the experiment. Ghrelin infusion increased plasma ghrelin levels 2.9-fold of baseline. Ghrelin significantly decreased LH pulse frequency (from 0.89 ± 0.07/h in baseline to 0.57 ± 0.10/h during ghrelin infusion; P < 0.05, mean ± sem), whereas LH pulse frequency remained unchanged during saline treatment. LH pulse amplitude was not affected. Ghrelin also significantly stimulated both cortisol and GH release, but had no effect on leptin. We conclude that ghrelin can inhibit GnRH pulse activity and may thereby mediate the suppression of the reproductive system observed in conditions of undernutrition, such as in anorexia nervosa. Ghrelin also activates the adrenal axis, but the relevance of this to the inhibition of GnRH pulse frequency remains to be established.

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Decrease in Luteinizing Hormone Pulse Frequency during a Five-Hour Peripheral Ghrelin Infusion in the Ovariectomized Rhesus Monkey

The Journal of Clinical Endocrinology & Metabolism Decrease in Luteinizing Hormone Pulse Frequency during a Five-Hour Peripheral Ghrelin Infusion in the Ovariectomized Rhesus Monkey NICOLAS R. VULLI E´MOZ 0 1 ENNIAN XIAO 0 1 LINNA XIA-ZHANG 0 1 MARC GERMOND 0 1 JEAN RIVIER 0 1 MICHEL FERIN 0 1 0 Abbreviations: AGRP , Agouti-related peptide; AVP, arginine vasopressin; GHS-R, GH secretagogue receptor; HPA, hypothalamic-pituitary-adrenal; NPY, neuropeptide Y; OVX, ovariectomized 1 Department of Obstetrics and Gynecology (N.R.V. , E.X., L.X.-Z., M.F.) , College of Physicians and Surgeons, Columbia University , New York , New York 10032; Reproductive Medicine Unit (M.G.), Department of Obstetrics and Gynecology, Centre Hospitalier Universitaire Vaudois , CH-1011 Lausanne , Switzerland; and The Salk Institute (J.R.) , La Jolla, California 92037 Ghrelin, a nutrition-related peptide secreted by the stomach, is elevated during prolonged food deprivation. Because undernutrition is often associated with a suppressed reproductive axis, we have postulated that increasing peripheral ghrelin levels will decrease the activity of the GnRH pulse generator. Adult ovariectomized rhesus monkeys (n 6) were subjected to a 5-h iv human ghrelin (100- to 150- g bolus followed by 100 -150 g/h) or saline infusion, preceded by a 3-h saline infusion to establish baseline pulsatile LH release. Blood samples were collected at 15-min intervals throughout the experiment. Ghrelin infusion increased plasma ghrelin levels 2.9-fold of baseline. Ghrelin significantly decreased LH - G HRELIN IS A 28-amino-acid peptide secreted primarily by the stomach. It was shown to be the endogenous ligand for the GH secretagogue receptor (GHS-R) ( 1 ), and indeed, injection of ghrelin induces a potent GH release in the rodent and in the human ( 2, 3 ). Studies in the rat and human also show that ghrelin strongly stimulates food intake ( 2, 4 –7 ). The arcuate nucleus is an important hypothalamic site in the control of food intake and has emerged as an integrative center for energy homeostasis, receiving neural signals from other areas of the hypothalamus and the brain as well as direct signals from energy-related hormones secreted peripherally ( 8, 9 ). Two potent orexigenic peptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), are colocated within arcuate neurons ( 10 ). NPY/AGRP neurons express GHS-R ( 11 ), and chronic central infusion of ghrelin increases hypothalamic NPY and AGRP mRNA levels in the rat ( 12 ), suggesting that the orexigenic effect of ghrelin is mediated by NPY and AGRP. In fact, in the double knockout mouse for NPY and AGRP, ablation of both peptides completely abolishes the orexigenic action of ghrelin ( 13 ). Plasma ghrelin levels are elevated during prolonged food deprivation ( 14 ), a condition associated with decreased reproductive axis activity ( 15 ). Moreover, high ghrelin levels JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. pulse frequency (from 0.89 0.07/h in baseline to 0.57 0.10/h during ghrelin infusion; P < 0.05, mean SEM), whereas LH pulse frequency remained unchanged during saline treatment. LH pulse amplitude was not affected. Ghrelin also significantly stimulated both cortisol and GH release, but had no effect on leptin. We conclude that ghrelin can inhibit GnRH pulse activity and may thereby mediate the suppression of the reproductive system observed in conditions of undernutrition, such as in anorexia nervosa. Ghrelin also activates the adrenal axis, but the relevance of this to the inhibition of GnRH pulse frequency remains to be established. (J Clin Endocrinol Metab 89: 5718 –5723, 2004) are reported in anorexia nervosa ( 16 ), a syndrome characterized by food restriction accompanied by amenorrhea and suppressed LH pulsatility as well as an activated adrenal axis ( 17 ). In these patients, ghrelin levels are normalized after partial weight recovery ( 16 ), suggesting that there may be a relationship between ghrelin increase due to malnutrition and reproductive dysfunction, because a return to normal body weight would also favor a normalization of reproductive function. These observations suggest that a prolonged plasma ghrelin elevation, mimicking a state of energy shortage, may suppress pulsatile LH release. The fact that ghrelin may exert its effect through the release of NPY and AGRP ( 13 ) and that these two neuropeptides have been shown to suppress pulsatile LH release in the ovariectomized (OVX) rhesus monkey ( 18, 19 ) support this hypothesis. Our goal here was to demonstrate that ghrelin can influence pulsatile LH release in the primate. For that purpose, we investigated the effects of a short-term increase in peripheral ghrelin levels, to levels that reflect undernutrition, on LH pulsatility. Materials and Methods Animals Six adult OVX rhesus monkeys (Macaca mulatta) weighing 5– 8 kg were used in the study. Ovariectomy had been performed at least 6 months before these experiments. The animals were housed in individual cages in temperature- and light-controlled rooms (lights on from 0800 –2000 h) and were fed twice a day with a high-protein Purina monkey chow (Purina Mills, St. Louis, MO) supplemented with fresh fruit or vegetables. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Columbia University. Experimental design Animals were briefly sedated with ketamine (5–7 mg/kg; Ketaset, Ford Dodge Animal Health, Ford Dodge, IA), and a catheter was inserted into the saphenous vein for blood sampling or hormone infusion. The animals were then seated in a primate chair to which they had previously been habituated. Each experimental protocol was started at 0745 h and lasted 8 h. After a 3-h period to document baseline hormone concentrations, animals were infused iv with saline (control) or ghrelin (human ghrelin, synthesized and chemically characterized in the laboratory of J.R.; 100- g bolus followed by 100 g/h) for a period of 5 h. In two unresponsive monkeys, the dose of ghrelin was increased to 150 g/h. Both animals responded to the higher dose, and the data of all six monkeys were pooled. Blood samples (1.2 ml) were obtained at 15-min intervals throughout the experiment for hormone measurements, and the same amount of saline was infused as replacement. Animals were not fed during the experiment. They were returned to their housing quarters at the end of the experiment and provided with food. Each experimental protocol was separated by at least 2 wk. Assays and data analysis Blood samples were centrifuged, and sera were kept at –20 C until assayed. LH was measured at 15-min intervals by a recombinant homologous RIA, as described previously ( 20 ), and using reagents provided by Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, Harbor-University of California Los Angeles Medical Center, Torrance, CA). Assay sensitivity (at 95% binding) was 0.06 ng/ml. Intra- and interassay coefficients of variation (CVs) were 6.0 and 15.7%, respectively. Cortisol and GH were measured at 45-min intervals using a commercial RIA kit for cortisol (Diagnostic System Laboratories Inc., Webster, TX) and a chemiluminescent immunoassay using the Immulite system (Diagnostic Products Corp. Inc., Los Angeles, CA) for GH. Intraassay CV for cortisol was 4.9%, interassay CVs were 2.5% for cortisol and 5.0% for GH. Ghrelin was measured hourly (in a pool made from four 15-min samples) throughout the experiment and measured with a RIA kit for total ghrelin (Linco Research Inc., St. Charles, MO) to determine ghrelin levels attained with the infusion. Baseline and 5-h sera were also measured for leptin using a RIA kit for primate leptin (Linco). All measurements for ghrelin and leptin were carried out with one assay; intra-assay CV’s were 6.5% for ghrelin and 3.9% for leptin. LH pulses were identified using three criteria for the analysis of LH pulsatility, as described previously ( 21, 22 ): first, the peak of LH must occur within 30 min of the previous nadir; second, the LH increase must exceed three times the LH intraassay CV; and third, the LH increase must be followed by declining levels in accord with the LH half-life. This analysis was then verified by the Cluster pulse-detection algorithm program (23); cluster sizes for peak and nadirs were set at 1 and 2, and to identify the increase and decrease of LH the t statistic used was 2.6. LH pulse frequency, LH pulse amplitude, and mean LH concentration were calculated; differences between the 3-h baseline and treatment and differences between saline and ghrelin administration were compared by the Student’s t test. Changes in cortisol and GH during saline and ghrelin administration were compared with baseline levels by ANOVA with repeated measures followed by Dunnett test. Areas under the cortisol and GH curves were calculated, and the responses to saline and ghrelin treatment were compared by the Student’s t test. Leptin levels by hour 5 of saline or ghrelin administration were compared with baseline by the Student’s t test. Results Ghrelin administration significantly decreased LH pulse frequency compared with either the baseline period or the saline control (Fig. 1). Table 1 summarizes LH pulse frequency, pulse amplitude, and concentration after the saline control and ghrelin infusion. Ghrelin stimulated the release of cortisol, which reached 125.5 7.6% (mean sem) of the baseline (P 0.05) 45 min after ghrelin injection (Fig. 2). Cortisol concentration decreased during the saline infusion, in accord with its circadian rhythm. This decrease became significant 90 min after the 3-h morning baseline, and by 5 h of the saline infusion cortisol concentration was 68.9 6.9% of the baseline. Mean areas under the cortisol curves in response to ghrelin administration were significantly higher then saline administration. Figure 3 illustrates LH and cortisol responses to saline and ghrelin administration in two individual monkeys. Ghrelin also stimulated GH levels that were significantly increased over baseline 45 and 90 min after ghrelin administration, although remaining unchanged during the saline experiment (Fig. 4). Mean areas under the GH curves in response to ghrelin administration were significantly higher than after the saline infusion. Ghrelin administration did not significantly affect leptin levels [baseline, 3.04 0.82 ng/ml (0.24 0.07 nmol/liter); 5 h after ghrelin, 2.89 1.17 ng/ml (0.23 0.09 nmol/liter)]. They remained unchanged after saline as well [baseline, 3.57 1.17 ng/ml (0.28 0.09 nmol/liter); 5 h after saline, 3.61 0.97 ng/ml (0.29 0.08 nmol/liter). Baseline ghrelin levels were similar in control and treatment groups and remained unchanged during the saline infusion. Ghrelin infusion significantly increased plasma ghrelin levels, which by 5 h had increased to a mean of 2.9-fold of baseline (baseline, 972 89.2 pg/ml; treatment, 2598 423.8). Figure 5 illustrates mean hourly ghrelin concentrations that were effective in inhibiting LH pulse frequency. Discussion Our data show for the first time in the nonhuman primate that peripheral administration of ghrelin clearly modulates the GnRH pulse generator, as demonstrated by a significant decrease in LH pulse frequency. The inhibitory effect on pulsatile LH release is observed while plasma ghrelin levels progressively increase over the course of a 5-h infusion to approximately 2.9 times baseline levels. The data also show that ghrelin stimulates both cortisol and GH release, whereas it has no effect on leptin secretion. The pathways by which peripherally secreted ghrelin may affect centrally located GnRH neurons remain to be fully understood. However, there is considerable circumstantial n 6; four animals received 100 g/h, and two animals received 150 g/h. a P 0.05 ghrelin treatment vs. saline treatment. b P 0.05 ghrelin treatment vs. baseline. FIG. 2. Mean ( SEM) cortisol responses to saline (E) and ghrelin (F) iv administration (n 6; four animals received 100 g/h, and two animals received 150 g/h). *, P 0.05 vs. baseline (ANOVA with repeated measures followed by Dunnett test); 0 baseline (mean levels of the 3-h baseline period). For conversion from g/dl to nmol/liter, multiply by 27.59. evidence to suggest that NPY and AGRP may mediate the inhibitory effect of ghrelin on the GnRH pulse generator. NPY and AGRP are colocated within the same neurons of the hypothalamic arcuate nucleus ( 10 ), an area that is viewed as an important site in the control of food intake ( 8, 9 ), and ghrelin administration in the rodent increases the synthesis of both NPY and AGRP (12). Both peptides exert a powerful orexigenic effect when infused centrally in the rodent and the rhesus monkey ( 24 –27 ), and the orexigenic action of ghrelin is abolished in double knockout mice for NPY and AGRP (13). Significantly, AGRP and NPY are up-regulated by food restriction ( 10 ), a condition known to suppress pulsatile LH release in the human and other species ( 28 –30 ) Finally, data in the OVX monkey have shown that NPY or AGRP infusions into the third ventricle inhibit pulsatile LH release ( 18, 19 ). Ghrelin has been previously reported to stimulate ACTH and glucocorticoid release in the rodent and in the human ( 31, 32 ). Our results in the monkey confirm this effect on the hypothalamic-pituitary-adrenal (HPA) axis: cortisol levels remain significantly elevated throughout the 5-h ghrelin infusion in comparison with the cortisol decline in the saline control, which reflects the expected circadian rhythm. Whether a longer-term ghrelin infusion would maintain elevated cortisol levels remains to be determined. Ghrelin has been shown to stimulate the release of both CRH and arginine vasopressin (AVP) from rat hypothalamic explants in vitro (31). Whether the in vivo data reflect a direct action of ghrelin on CRH and AVP release or whether effects of ghrelin on the HPA axis are also mediated by NPY and AGRP remains to be demonstrated. However, we believe the latter is likely because both neuropeptides are known to activate ACTH and cortisol release in the rhesus monkey when administered centrally ( 25, 33 ). Whether there is a causal relationship between the activation of the HPA axis by ghrelin and the decrease in LH pulse frequency remains to be investigated. That this is possible is reflected by observations of a primary mediatory role of central HPA pathways in the control of the reproductive axis during stress in the primate: exogenous administration of CRH or AVP results in a rapid decline in pulsatile LH release ( 22, 34 ), whereas antagonism of endogenous CRH or AVP activity prevents the decrease in pulsatile LH release that follows a stress challenge ( 35, 36 ). Our finding substantiates a report in the OVX estradioltreated rat showing that administration of ghrelin into the third ventricle rapidly suppresses pulsatile LH secretion ( 37 ). However, although our animals had been OVX, they did not receive estrogen replacement. Reports in healthy men after a single bolus of ghrelin ( 32 ) or in the male rat after central or ip ghrelin administration were unable to demonstrate a significant effect on plasma LH release ( 2 ). Whether these negative data reflect gender differences or other experimental conditions is unknown. The decrease in LH pulse frequency observed after ghrelin, although modest in our short-term experimental paradigm, is, we believe, an important observation in regard to a potential relationship between energy-related peptides and the reproductive cycle. Indeed, it is known that optimal activity of the GnRH pulse generator is essential for a functional reproductive system and that in monkeys bearing lesions of the arcuate nucleus and replaced with pulses of GnRH, abnormalities of menstrual cyclicity are readily observed if GnRH pulse frequency is reduced from one pulse/60 min to one pulse/90 min ( 38 ). Significantly, Loucks and Thuma ( 28 ) have recently quantified the relationship between energy availability and changes in LH pulsatility in a group of regularly cycling sedentary women and have shown a surprisingly abrupt decrease in LH pulse frequency at a specific threshold of energy availability. In this study, at 22% of normal caloric intake, the mean number of LH pulses per 24 h decreased by 5.8 1.6 pulses, a number similar to that after ghrelin when compared for an equivalent time period. Plasma ghrelin levels in patients with anorexia nervosa ( 16 ) and in women after physical exercise ( 39 ) are elevated compared with age-matched controls. Data in amenorrheic women have shown a significant decrease in LH pulse frequency ( 40 – 42 ), which according to our data may possibly be related to the amplified ghrelin signal, although this remains to be demonstrated. The decrease in LH pulse frequency after short-term ghrelin infusion that we report here was obtained at ghrelin levels comparable to those reported in these patients. Additional studies will investigate whether a longer ghrelin FIG. 4. Mean ( SEM) GH responses to saline (E) and ghrelin (F) iv administration (n 6; four animals received 100 g/h, and two animals received 150 g/h). *, P 0.05 vs. baseline (ANOVA with repeated measures followed by Dunnett test); 0 baseline (mean levels of the 3-h baseline period). For conversion from ng/ml to mIU/liter, multiply by 2.6. infusion can produce a more sustained and/or more profound inhibition of the GnRH pulse generator. Intriguingly, cortisol concentrations are elevated in patients with anorexia nervosa ( 43 ) and in women in whom energy availability is decreased ( 28 ). Although a primary role of ghrelin on the reproductive FIG. 5. Mean ( SEM) ghrelin level during the 3-h baseline period (B) and at hourly intervals (H1–H5) during the saline and ghrelin iv infusion (n 6; four animals received 100 g/h, and two animals received 150 g/h). *, P 0.05 vs. baseline (ANOVA with repeated measures followed by Dunnett test) and 5-h saline infusion (Student’s t test). For conversion from pg/ml to pmol/liter, multiply by 0.296. system is at first view not supported by the observation that ghrelin-null mice display normal responses to starvation and have a normal reproductive function ( 44 ), it is also probable that deletion of ghrelin during early life may activate compensatory pathways. Ghrelin acutely stimulates GH release in the monkey, although high GH levels were not maintained throughout the 5-h ghrelin infusion. A GH release was predicted from observations that ghrelin is an endogenous ligand for the GHS-R ( 1 ) and from previous data in the rat and human ( 2, 3, 32 ). Leptin levels remain unchanged after ghrelin infusion. Ghrelin and leptin are two peripherally secreted peptides acting centrally to regulate energy homeostasis by exerting antagonistic actions on food intake and energy expenditure ( 45 ), possibly part of an integrated system in which these peptides counterbalance each other. Our data and others showing that ghrelin levels are not regulated by leptin administration in healthy subjects ( 46 ) do not support this contention, at least within the limited period of our experiment. In conclusion, we have shown that a short-term peripheral ghrelin infusion, which elevates plasma ghrelin levels 2.9fold of baseline, significantly decreases LH pulse frequency in the OVX rhesus monkey, suggesting an inhibitory effect of ghrelin on the GnRH pulse generator. The data indicate that ghrelin may be one of the factors mediating the inhibitory effects of a negative energy balance on reproductive function. Additional studies will focus on the long-term effects of ghrelin on reproductive function as well as on the neuropeptides involved in the process. Acknowledgments We acknowledge Laura Cervini for her help in the synthesis of ghrelin and Alinda Barth and Nancy Cotui for their help in hormonal assays. Reagents for monkey LH RIA were obtained through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, with the help of Dr. A. F. Parlow. Received June 29, 2004. Accepted August 16, 2004. Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, College of Physicians & Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: . This work was supported by National Institutes of Health Grants RO1-HD-46715 and RO1-DK-39144, by Grant R01-HD-039899 to J.R., and by fellowships grants to N.R.V. from Novartis Stiftung, vormals Ciba-Geigy-Jubila¨ ums-Stiftung, and Socie´te´ Acade´mique Vaudoise. JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 1. 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Vulliémoz, Nicolas R., Xiao, Ennian, Xia-Zhang, Linna, Germond, Marc, Rivier, Jean, Ferin, Michel. Decrease in Luteinizing Hormone Pulse Frequency during a Five-Hour Peripheral Ghrelin Infusion in the Ovariectomized Rhesus Monkey, The Journal of Clinical Endocrinology & Metabolism, 2004, 5718-5723, DOI: 10.1210/jc.2004-1244