Central Infusion of Agouti-Related Peptide Suppresses Pulsatile Luteinizing Hormone Release in the Ovariectomized Rhesus Monkey
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内分泌学杂志 2005年第2期
Departments of Obstetrics and Gynecology (N.R.V., E.X., L.X.-Z., M.F.) and Medicine (S.L.W.), College of Physicians and Surgeons, Columbia University, New York, New York 10032
Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: mf8@columbia.edu.
Abstract
Agouti-related peptide (AGRP), an endogenous melanocortin receptor antagonist, is a powerful orexigenic peptide when infused centrally. AGRP and neuropeptide Y (NPY), another orexigenic peptide, are colocated within the same neurons in the arcuate nucleus. Both NPY and AGRP mRNA expression increases during food restriction, a condition that is known to suppress the GnRH pulse generator and reproductive function. Although NPY has been shown previously to suppress LH secretion in the ovariectomized monkey, data on AGRP are lacking. In this study, we examined the effect of AGRP infusion into the third ventricle on pulsatile LH release in five adult monkeys. The 8-h protocol included a 3-h intraventricular saline infusion to establish baseline pulsatile LH release, followed by a 5-h infusion of AGRP (83–132) [5 μg/h (n = 1) or 10 μg/h (n = 4)]. In separate experiments, each animal received an 8-h saline treatment as a control. Blood samples were collected every 15 min for LH measurements. Cortisol levels were measured every 45 min. AGRP infusion significantly decreased LH pulse frequency (from a baseline of 0.74 ± 0.07 pulse/h to 0.36 ± 0.12 during AGRP infusion; P < 0.01) and mean LH concentrations (to 41.1 ± 7.5% of baseline by h 5 of AGRP infusion; P < 0.001). LH pulse amplitude was not modified by AGRP treatment. AGRP infusion also significantly increased cortisol release, as previously reported. The data demonstrate that central administration of AGRP inhibits pulsatile LH release in the monkey and suggest that AGRP, like NPY, may mediate the effect of a negative energy balance on the reproductive system by suppressing the GnRH pulse generator.
Introduction
THERE IS GOOD clinical evidence in the primate linking energy homeostasis and reproductive function (1, 2, 3). Nutritional deprivation and abnormal eating habits are known to interfere with the normal reproductive process, and a functional reproductive system requires an accurate integration of energy balance (2, 4, 5). Whereas the inhibitory effects of negative energy balance on the hypothalamic-pituitary-gonadal axis are known, the central mechanisms whereby the reproductive axis is affected remain to be investigated. The discovery of peptides involved in the control of food intake has greatly advanced our understanding of energy homeostasis. Agouti-related peptide (AGRP), the endogenous melanocortin receptor (MC3-R and MC4-R) antagonist (6), and neuropeptide Y (NPY) are two powerful orexigenic neuropeptides, which when infused centrally increase food intake in the rodent and monkey (7, 8, 9, 10). There is considerable anatomical and functional overlap between these two neuropeptides. NPY and AGRP are colocalized within the same neurons in the arcuate nucleus of the hypothalamus (11). Both mRNAs are up-regulated in situations of energy shortage, conditions that are known to suppress the reproductive axis (11, 12, 13).
NPY has been shown to have different effects on pulsatile GnRH/LH activity, depending on the pattern of NPY administration and steroid hormone milieu. A continuous NPY infusion into the third ventricle decreases pulsatile activity of GnRH/LH in the ovariectomized (OVX) monkey (14) and rat (15). A stimulatory effect of AGRP on pulsatile LH release was shown in the intact male rat (16). There are currently no data in the primate on the effect of AGRP on pulsatile LH release. The anatomical overlap of AGRP and NPY and their similar functional effects on nutrition also suggest a role for AGRP in mediating the inhibitory effects of undernutrition on the reproductive axis. As a first step toward clarifying such a role for AGRP, we examined effects of a continuous AGRP infusion into the third ventricle on pulsatile LH release in the OVX rhesus monkey, using the same experimental conditions under which NPY was shown to inhibit pulsatile LH activity (14).
Materials and Methods
Animals
Five adult female rhesus monkeys (Macaca mulatta) weighing 5–7.5 kg were used in these experiments. Monkeys were housed in individual cages in temperature- (19–22 C) and light-controlled rooms (lights on 0700–1900 h). They were fed twice a day with high-protein Purina monkey chow (Purina Mills, St. Louis, MO), supplemented with fresh fruits or vegetables. Water was available at all times. Each animal had been ovariectomized at least 3 months before the study. For intraventricular peptide infusion, a chronic cannula was implanted stereotaxically into the third ventricle. Animals were sedated with ketamine (Ketaset 5–7 mg/kg, Henry Schein Inc., Melville, NY) and intubated. Gas anesthesia (isoflurane 1.5–2.0% supplemented by oxygen) was then initiated. The posterior clinoid was visualized by fluoroscopy on lateral pictures and chosen as the target for the tip of the cannula, thereby determining the anterior-posterior coordinate. A burr hole (0.5 cm in diameter) was drilled on the midline until clear visualization of the sagittal sinus. A cannula (18 gauge, 37–40 mm in length) was then inserted through the sagittal sinus and advanced to a depth of 18–20 mm from the dura until the top of the third ventricle was reached, as confirmed by consistent cerebrospinal fluid dripping. The cannula was then secured to the skull with dental acrylic cement and protected by a plastic cap with a screw-off top anchored to the calvarium. For intraventricular infusions, a Silastic tubing was inserted into the cannula 8–15 mm beyond its tip to reach the bottom of the third ventricle. The intraventricular Silastic tubing was then connected to a Hamilton syringe attached to an infusion pump. All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Experimental design
Experimental protocols were performed on animals seated in a primate chair, as previously described (17). The night before each experiment, animals were briefly sedated with 5–7 mg/kg ketamine, and a catheter was inserted into a saphenous vein for blood collection. Each monkey had previously been adapted to this procedure. The experiment started on the next morning at 0800 h. After a 3-h physiological saline intraventricular infusion (25 μl/h) to document hormone baseline, human AGRP (83–132)-NH2 (Phoenix Pharmaceuticals, Inc., Belmont, CA) were then infused for 5 h. AGRP was dissolved in saline and the infusion rate was kept at 25 μl/h. To determine the effective dose of AGRP capable of inhibiting pulsatile LH release, AGRP doses of 1–20 μg/h were tested in the first three monkeys. One of three animals responded to 5 μg/h (monkey 8YL), whereas two of two responded to 10 μg/h (8YL could not be tested with the 10-μg dose for technical reasons). The 10-μg dose was then used in the remaining two animals. As a control, each monkey also received an 8-h infusion of saline (25 μl/h). Blood samples (1.2 ml) were taken at 15-min intervals. Animals were fed fresh fruits during the 8-h protocol and returned to their own cage at the end of each protocol. Each experimental protocol was separated by at least 2 wk.
Hormone assays
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 (18), 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 was measured at 45-min intervals with a commercial RIA kit for cortisol (Diagnostic System Laboratories Inc., Webster, TX). Intra- and interassay CVs were 4.9 and 2.5%, respectively.
Data analysis and statistics
Hormonal data were expressed as mean ± SEM. Statistical analysis was performed using PRISM (GraphPad, San Diego, CA). Hourly areas under the LH curve after AGRP or saline treatment were calculated as a percentage of the 3-h baseline and analyzed by the Kruskal-Wallis ranking test followed by the Dunn test. For this calculation, the results of four monkeys having received the 10-μg/h dose and one animal (8YL) at 5 μg/h were pooled because both the LH and cortisol responses to AGRP were similar. LH pulse analysis was performed as described previously (19), using three criteria to identify LH pulses: the difference between peak and previous nadir must exceed three times the intraassay CV; the peak must occur within 30 min of the nadir, and the LH peak must be followed by a decrease in accord with the half-life of LH. LH pulse frequency, pulse amplitude, and mean concentration were calculated, and the difference between AGRP and saline treatment was analyzed by paired t test. Areas under the cortisol curves were calculated and the cortisol responses to AGRP or saline were compared by paired t test.
Results
Infusion of AGRP into the third ventricle significantly suppressed pulsatile LH release. Whereas mean LH pulse frequency remained unchanged after saline infusion, it decreased from 0.74 ± 0.07 pulse/h during the 3-h baseline to 0.36 ± 0.12 during the 5-h AGRP infusion (P < 0.01 vs. baseline and saline control) (Fig. 1). AGRP infusion significantly decreased mean LH levels as well, but the individual LH response profile to AGRP varied. In two of five monkeys, the decrease in LH occurred within the first 2 h after AGRP, whereas in the two other animals, a decrease occurred only by h 4. Overall, mean LH concentrations in these five animals were significantly decreased by h 4 of the AGRP infusion, and by h 5 the mean percentage area under the LH curve was 41.1 ± 7.5% of baseline, compared with 94.9 ± 9.7% after saline (P < 0.001) (Fig. 2). Mean LH pulse amplitude remained unchanged. Whereas cortisol concentrations decreased during saline treatment (baseline: 28.0 ± 1.84; h 5: 19.4 ± 2.9 μg/dl), reflecting the normal circadian rhythm, this decrease in cortisol was prevented by AGRP treatment (baseline: 29.3 ± 1.3; h 5: 26.0 ± 2.8 μg/dl). The mean area under the cortisol curve therefore increased to 7678 ± 683 μg/dl during 5-h AGRP infusion vs. 5614 ± 510.5 during saline infusion (P < 0.05) (Fig. 3). Figure 4 illustrates pulsatile LH release and cortisol changes during infusions of saline and AGRP into the third ventricle in two individual monkeys having received 10 μg/h. Figure 5 shows LH and cortisol responses to saline and AGRP 5 μg/h infusion in monkey 8YL. These responses are similar to those observed in the other four monkeys having received 10 μg/h.
FIG. 1. LH pulse frequency is significantly decreased during AGRP infusion into the third ventricle, compared with baseline, and to a 5-h saline treatment (**, P < 0.01). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL).
FIG. 2. LH concentrations, calculated as hourly areas under the LH curve and expressed as a percentage of the 3-h mean baseline, are significantly reduced by AGRP (**, P < 0.01 vs. baseline; ***, P < 0.001 vs. baseline). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL). LH concentrations remained unchanged during saline infusion. 0, Mean baseline as calculated from the first 3 h.
FIG. 3. Cortisol concentrations, shown as mean hourly areas under the cortisol curve during saline and AGRP infusions. The mean overall area during the 5-h AGRP infusion was significantly greater than that during the saline infusion (P < 0.05). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL). 0, Mean baseline as calculated from the first 3 h.
FIG. 4. LH (closed circles) and cortisol (open squares) responses to saline (left panels) and AGRP (right panels) infusions into the third ventricle in two monkeys. *, LH pulse. The vertical line indicates the end of the 3-h baseline and the start of the 5-h treatment (saline or AGRP) period.
FIG. 5. LH (closed circles) and cortisol (open squares) responses to saline (left panels) and AGRP (5 μg/h) (right panel) infusions into the third ventricle in monkey 8YL. This dose was as effective as the 10-μg/h dose used in the other four monkeys. *, LH pulse. The vertical line indicates the end of the 3-h baseline and the start of the 5-h treatment (saline or AGRP) period
Discussion
Our results demonstrate, for the first time in the primate, an inhibitory effect of AGRP on pulsatile LH release. AGRP infusion into the third ventricle significantly suppressed mean LH plasma levels and decreased LH pulse frequency in OVX rhesus monkeys. The results support a central action of this orexigenic neuropeptide on the GnRH pulse generator in the hypothalamic arcuate nucleus and suggest that AGRP plays a role in the control of the reproductive axis in the primate. Pilot experiments in which AGRP was infused into the lateral ventricle (not reported here) were less successful, in that only two of five monkeys responded with a decrease in LH highlighting the importance of a central infusion site in the proximity of the arcuate nucleus of the hypothalamus. Even though pulsatile LH release was inhibited by infusion of AGRP into the third ventricle in all five tested monkeys, the response profile was different: the LH decrease was rapid in three animals but slower in the others, presumably reflecting varying locations of the catheter tip in relation to the arcuate nucleus.
Our data on the inhibitory effects of AGRP on pulsatile LH release are concordant with results obtained after NPY infusions, as well as into the third ventricle, in OVX rhesus monkeys. Similarly to AGRP, NPY reduced LH pulse frequency and mean LH levels, also indicating an inhibition of pulsatile GnRH-LH release (14). Taken together, the data from both experiments provide convincing support for a role of these two orexigenic neuropeptides in the control of the GnRH pulse generator. Significantly, AGRP and NPY are synthesized within the same neurons of the arcuate nucleus (11), which in the primate is also the presumed site of the GnRH pulse generator (20). Expression of both neuropeptides is increased with negative energy balance (11, 12, 13), a situation known to suppress the reproductive axis (1, 2, 3). The mechanisms by which NPY and AGRP influence energy homeostasis are, however, different. NPY acts through the activation of Y1 and Y5 receptors (9, 21, 22), whereas AGRP is an endogenous antagonist of MSH at MC3-R and MC4-R (6). Whether similar pathways mediate the effect of these two neuropeptides on the GnRH pulse generator remains to be fully investigated. However, suppressive effects of NPY on LH secretion in the rodent have been shown to be mediated by NPY Y1 and Y5 receptors (23, 24, 25). The mediatory role of MC3-R and/or MC4-R in the inhibitory effect of AGRP on pulsatile LH release is not known. Of interest are studies in the rodent that suggest that effects of a single central injection of AGRP on food intake are very potent (26, 27, 28), whereas NPY, even at very high dose, has little or no effect on food intake beyond 24 h (29). These differential effects of AGRP and NPY on food intake may reflect their selective pathways.
The observation that LH pulse frequency is decreased after AGRP administration suggests that this inhibitory effect of AGRP on the hypothalamic-pituitary-gonadal axis is mediated centrally through suppression of the GnRH pulse generator. Several pathways may be postulated to mediate this effect of AGRP. First, morphological data show that NPY/AGRP neurons in the arcuate nucleus have direct axonal contacts on the somata and proximal dendrites of GnRH neurons (30), suggesting that there may be a direct inhibitory effect of AGRP on the GnRH neurons. However, the type and location of receptors involved in this synaptic interaction are currently unknown.
Second, AGRP may suppress GnRH pulsatile release by enhancing the inhibitory effects of ?-endorphin (?-EP) on the GnRH neuron. ?-EP is derived from proopiomelanocortin, the same precursor that yields MSH, and proopiomelanocortin neurons are also known to contact GnRH neurons in the rodent (31). ?-EP is a strong inhibitor of the GnRH pulse generator, and we previously demonstrated that MSH can counteract the inhibitory effect of ?-EP on pulsatile LH release, suggesting that the ?-EP to MSH ratio is important in the control of pulsatile GnRH release (32). These data suggest that, because AGRP is an endogenous melanocortin receptor antagonist (6), elevations in AGRP levels may suppress pulsatile GnRH release by antagonizing endogenous MSH activity, thereby enhancing the inhibitory action of ?-EP.
Third, AGRP may suppress the GnRH pulse generator through activation of hypothalamus-pituitary-adrenal (HPA) pathways involved in the gonadotropin response to stressors because our present data also show that third-ventricle AGRP infusion in the OVX monkey increases cortisol release, confirming previous results (17). NPY has also been shown to stimulate the HPA endocrine axis in the intact male rhesus monkey and the dog (9, 33). Arcuate NPY/AGRP neurons project to the paraventricular nucleus, the location of CRH/AVP-containing neurons (34, 35). AGRP has been shown to increase the release of CRH and AVP, the two major stress-related neuropeptides, from hypothalamic explants (36). These HPA neuropeptides play a primary role in the control of pulsatile LH release in stress (37): administration of CRH or AVP results in a rapid decline in pulsatile LH release (18, 38), whereas inactivation of endogenous CRH or AVP activity by antagonists prevents the inhibition of pulsatile LH decrease that follows stressors (39, 40).
Fourth, AGRP may also directly activate opioid pathways and thereby inhibit pulsatile LH release. Studies have shown that the acute stimulatory effect of NPY and AGRP on food intake is mediated by μ- and -opiate receptors because administration of the nonspecific opioid receptor antagonist naloxone blocks this effect (41, 42). In the arcuate nucleus, NPY/AGRP neurons are synaptically linked to ?-EP-containing cells (43). These four putative pathways whereby AGRP may modulate pulsatile LH release remain to be fully investigated and may not be mutually exclusive.
Our experiments with AGRP showing an inhibition of pulsatile LH release, as those with NPY (14), were performed in the OVX monkey. In contrast, in intact male rats, intracerebroventricular administration of AGRP was shown to acutely increase basic pulsatile LH secretion (16). This difference in response may be species related or reflect the steroid environment. Indeed, effects of NPY on gonadotropin release are markedly influenced by the steroid status. For instance, whereas NPY inhibits pulsatile LH release in the castrated rat, sheep, rabbit, and monkey (14, 15, 44, 45), this peptide stimulates LH release in the intact male rat (46) and steroid-pretreated OVX rat (47). In the OVX-replaced monkey, however, estradiol appears to desensitize the animal to the inhibitory effect of NPY (14). Whether and how the steroid environment influences the LH response to AGRP remains to be investigated. It should also be mentioned that the mode of administration and/or site of AGRP or NPY infusion may also influence the LH response. It has indeed been shown that a pulsatile NPY infusion into the stalk-median eminence area is stimulatory to pulsatile GnRH release in the OVX monkey (48).
In summary, we have demonstrated that infusion of AGRP into the third ventricle suppresses pulsatile LH release in the OVX rhesus monkey, a result identical with that reported for NPY. We postulate that both AGRP and NPY may mediate the inhibitory effects of ghrelin and/or negative energy balance on the GnRH pulse generator and the reproductive system. The result of our study is relevant to physiopathological situations in which negative energy balance is combined with reproductive dysfunction because both AGRP and NPY mRNAs are up-regulated in food-deprived animals (11). Ghrelin, a powerful orexigenic peptide secreted by the stomach (49, 50, 51), is also chronically elevated in anorexia nervosa patients (52). Interestingly, in the rodent the effect of ghrelin on food intake is mediated by the NPY/AGRP neurons (53). We also recently demonstrated that a peripheral infusion of ghrelin in the OVX rhesus monkey decreases LH pulse frequency, indicating that ghrelin can also decrease the activity of the GnRH pulse generator (54). Further studies will be necessary to determine whether the inhibitory effect of ghrelin on the GnRH pulse generator is also mediated by NPY and/or AGRP.
Acknowledgments
We thank Dr. D. Van Vugt for his technological advice in regard to the third ventricular cannulation. Reagents for the monkey LH RIA were obtained with the help of Dr. A. F. Parlow (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA).
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Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S 2004 Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 145:2607–2612
Vulliemoz NR, Xiao E, Xia-Zhang L, Germond M, Rivier J, Ferin M2004 Decrease in LH pulse frequency during a five-hour peripheral ghrelin infusion in the ovariectomized rhesus monkey. J Clin Endocrinol Metab 89:5718–5723(Nicolas R. Vulliémoz, Enn)
Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: mf8@columbia.edu.
Abstract
Agouti-related peptide (AGRP), an endogenous melanocortin receptor antagonist, is a powerful orexigenic peptide when infused centrally. AGRP and neuropeptide Y (NPY), another orexigenic peptide, are colocated within the same neurons in the arcuate nucleus. Both NPY and AGRP mRNA expression increases during food restriction, a condition that is known to suppress the GnRH pulse generator and reproductive function. Although NPY has been shown previously to suppress LH secretion in the ovariectomized monkey, data on AGRP are lacking. In this study, we examined the effect of AGRP infusion into the third ventricle on pulsatile LH release in five adult monkeys. The 8-h protocol included a 3-h intraventricular saline infusion to establish baseline pulsatile LH release, followed by a 5-h infusion of AGRP (83–132) [5 μg/h (n = 1) or 10 μg/h (n = 4)]. In separate experiments, each animal received an 8-h saline treatment as a control. Blood samples were collected every 15 min for LH measurements. Cortisol levels were measured every 45 min. AGRP infusion significantly decreased LH pulse frequency (from a baseline of 0.74 ± 0.07 pulse/h to 0.36 ± 0.12 during AGRP infusion; P < 0.01) and mean LH concentrations (to 41.1 ± 7.5% of baseline by h 5 of AGRP infusion; P < 0.001). LH pulse amplitude was not modified by AGRP treatment. AGRP infusion also significantly increased cortisol release, as previously reported. The data demonstrate that central administration of AGRP inhibits pulsatile LH release in the monkey and suggest that AGRP, like NPY, may mediate the effect of a negative energy balance on the reproductive system by suppressing the GnRH pulse generator.
Introduction
THERE IS GOOD clinical evidence in the primate linking energy homeostasis and reproductive function (1, 2, 3). Nutritional deprivation and abnormal eating habits are known to interfere with the normal reproductive process, and a functional reproductive system requires an accurate integration of energy balance (2, 4, 5). Whereas the inhibitory effects of negative energy balance on the hypothalamic-pituitary-gonadal axis are known, the central mechanisms whereby the reproductive axis is affected remain to be investigated. The discovery of peptides involved in the control of food intake has greatly advanced our understanding of energy homeostasis. Agouti-related peptide (AGRP), the endogenous melanocortin receptor (MC3-R and MC4-R) antagonist (6), and neuropeptide Y (NPY) are two powerful orexigenic neuropeptides, which when infused centrally increase food intake in the rodent and monkey (7, 8, 9, 10). There is considerable anatomical and functional overlap between these two neuropeptides. NPY and AGRP are colocalized within the same neurons in the arcuate nucleus of the hypothalamus (11). Both mRNAs are up-regulated in situations of energy shortage, conditions that are known to suppress the reproductive axis (11, 12, 13).
NPY has been shown to have different effects on pulsatile GnRH/LH activity, depending on the pattern of NPY administration and steroid hormone milieu. A continuous NPY infusion into the third ventricle decreases pulsatile activity of GnRH/LH in the ovariectomized (OVX) monkey (14) and rat (15). A stimulatory effect of AGRP on pulsatile LH release was shown in the intact male rat (16). There are currently no data in the primate on the effect of AGRP on pulsatile LH release. The anatomical overlap of AGRP and NPY and their similar functional effects on nutrition also suggest a role for AGRP in mediating the inhibitory effects of undernutrition on the reproductive axis. As a first step toward clarifying such a role for AGRP, we examined effects of a continuous AGRP infusion into the third ventricle on pulsatile LH release in the OVX rhesus monkey, using the same experimental conditions under which NPY was shown to inhibit pulsatile LH activity (14).
Materials and Methods
Animals
Five adult female rhesus monkeys (Macaca mulatta) weighing 5–7.5 kg were used in these experiments. Monkeys were housed in individual cages in temperature- (19–22 C) and light-controlled rooms (lights on 0700–1900 h). They were fed twice a day with high-protein Purina monkey chow (Purina Mills, St. Louis, MO), supplemented with fresh fruits or vegetables. Water was available at all times. Each animal had been ovariectomized at least 3 months before the study. For intraventricular peptide infusion, a chronic cannula was implanted stereotaxically into the third ventricle. Animals were sedated with ketamine (Ketaset 5–7 mg/kg, Henry Schein Inc., Melville, NY) and intubated. Gas anesthesia (isoflurane 1.5–2.0% supplemented by oxygen) was then initiated. The posterior clinoid was visualized by fluoroscopy on lateral pictures and chosen as the target for the tip of the cannula, thereby determining the anterior-posterior coordinate. A burr hole (0.5 cm in diameter) was drilled on the midline until clear visualization of the sagittal sinus. A cannula (18 gauge, 37–40 mm in length) was then inserted through the sagittal sinus and advanced to a depth of 18–20 mm from the dura until the top of the third ventricle was reached, as confirmed by consistent cerebrospinal fluid dripping. The cannula was then secured to the skull with dental acrylic cement and protected by a plastic cap with a screw-off top anchored to the calvarium. For intraventricular infusions, a Silastic tubing was inserted into the cannula 8–15 mm beyond its tip to reach the bottom of the third ventricle. The intraventricular Silastic tubing was then connected to a Hamilton syringe attached to an infusion pump. All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Experimental design
Experimental protocols were performed on animals seated in a primate chair, as previously described (17). The night before each experiment, animals were briefly sedated with 5–7 mg/kg ketamine, and a catheter was inserted into a saphenous vein for blood collection. Each monkey had previously been adapted to this procedure. The experiment started on the next morning at 0800 h. After a 3-h physiological saline intraventricular infusion (25 μl/h) to document hormone baseline, human AGRP (83–132)-NH2 (Phoenix Pharmaceuticals, Inc., Belmont, CA) were then infused for 5 h. AGRP was dissolved in saline and the infusion rate was kept at 25 μl/h. To determine the effective dose of AGRP capable of inhibiting pulsatile LH release, AGRP doses of 1–20 μg/h were tested in the first three monkeys. One of three animals responded to 5 μg/h (monkey 8YL), whereas two of two responded to 10 μg/h (8YL could not be tested with the 10-μg dose for technical reasons). The 10-μg dose was then used in the remaining two animals. As a control, each monkey also received an 8-h infusion of saline (25 μl/h). Blood samples (1.2 ml) were taken at 15-min intervals. Animals were fed fresh fruits during the 8-h protocol and returned to their own cage at the end of each protocol. Each experimental protocol was separated by at least 2 wk.
Hormone assays
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 (18), 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 was measured at 45-min intervals with a commercial RIA kit for cortisol (Diagnostic System Laboratories Inc., Webster, TX). Intra- and interassay CVs were 4.9 and 2.5%, respectively.
Data analysis and statistics
Hormonal data were expressed as mean ± SEM. Statistical analysis was performed using PRISM (GraphPad, San Diego, CA). Hourly areas under the LH curve after AGRP or saline treatment were calculated as a percentage of the 3-h baseline and analyzed by the Kruskal-Wallis ranking test followed by the Dunn test. For this calculation, the results of four monkeys having received the 10-μg/h dose and one animal (8YL) at 5 μg/h were pooled because both the LH and cortisol responses to AGRP were similar. LH pulse analysis was performed as described previously (19), using three criteria to identify LH pulses: the difference between peak and previous nadir must exceed three times the intraassay CV; the peak must occur within 30 min of the nadir, and the LH peak must be followed by a decrease in accord with the half-life of LH. LH pulse frequency, pulse amplitude, and mean concentration were calculated, and the difference between AGRP and saline treatment was analyzed by paired t test. Areas under the cortisol curves were calculated and the cortisol responses to AGRP or saline were compared by paired t test.
Results
Infusion of AGRP into the third ventricle significantly suppressed pulsatile LH release. Whereas mean LH pulse frequency remained unchanged after saline infusion, it decreased from 0.74 ± 0.07 pulse/h during the 3-h baseline to 0.36 ± 0.12 during the 5-h AGRP infusion (P < 0.01 vs. baseline and saline control) (Fig. 1). AGRP infusion significantly decreased mean LH levels as well, but the individual LH response profile to AGRP varied. In two of five monkeys, the decrease in LH occurred within the first 2 h after AGRP, whereas in the two other animals, a decrease occurred only by h 4. Overall, mean LH concentrations in these five animals were significantly decreased by h 4 of the AGRP infusion, and by h 5 the mean percentage area under the LH curve was 41.1 ± 7.5% of baseline, compared with 94.9 ± 9.7% after saline (P < 0.001) (Fig. 2). Mean LH pulse amplitude remained unchanged. Whereas cortisol concentrations decreased during saline treatment (baseline: 28.0 ± 1.84; h 5: 19.4 ± 2.9 μg/dl), reflecting the normal circadian rhythm, this decrease in cortisol was prevented by AGRP treatment (baseline: 29.3 ± 1.3; h 5: 26.0 ± 2.8 μg/dl). The mean area under the cortisol curve therefore increased to 7678 ± 683 μg/dl during 5-h AGRP infusion vs. 5614 ± 510.5 during saline infusion (P < 0.05) (Fig. 3). Figure 4 illustrates pulsatile LH release and cortisol changes during infusions of saline and AGRP into the third ventricle in two individual monkeys having received 10 μg/h. Figure 5 shows LH and cortisol responses to saline and AGRP 5 μg/h infusion in monkey 8YL. These responses are similar to those observed in the other four monkeys having received 10 μg/h.
FIG. 1. LH pulse frequency is significantly decreased during AGRP infusion into the third ventricle, compared with baseline, and to a 5-h saline treatment (**, P < 0.01). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL).
FIG. 2. LH concentrations, calculated as hourly areas under the LH curve and expressed as a percentage of the 3-h mean baseline, are significantly reduced by AGRP (**, P < 0.01 vs. baseline; ***, P < 0.001 vs. baseline). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL). LH concentrations remained unchanged during saline infusion. 0, Mean baseline as calculated from the first 3 h.
FIG. 3. Cortisol concentrations, shown as mean hourly areas under the cortisol curve during saline and AGRP infusions. The mean overall area during the 5-h AGRP infusion was significantly greater than that during the saline infusion (P < 0.05). AGRP dose (n = 5): 10 μg/h in four monkeys and 5 μg/h in one monkey (8YL). 0, Mean baseline as calculated from the first 3 h.
FIG. 4. LH (closed circles) and cortisol (open squares) responses to saline (left panels) and AGRP (right panels) infusions into the third ventricle in two monkeys. *, LH pulse. The vertical line indicates the end of the 3-h baseline and the start of the 5-h treatment (saline or AGRP) period.
FIG. 5. LH (closed circles) and cortisol (open squares) responses to saline (left panels) and AGRP (5 μg/h) (right panel) infusions into the third ventricle in monkey 8YL. This dose was as effective as the 10-μg/h dose used in the other four monkeys. *, LH pulse. The vertical line indicates the end of the 3-h baseline and the start of the 5-h treatment (saline or AGRP) period
Discussion
Our results demonstrate, for the first time in the primate, an inhibitory effect of AGRP on pulsatile LH release. AGRP infusion into the third ventricle significantly suppressed mean LH plasma levels and decreased LH pulse frequency in OVX rhesus monkeys. The results support a central action of this orexigenic neuropeptide on the GnRH pulse generator in the hypothalamic arcuate nucleus and suggest that AGRP plays a role in the control of the reproductive axis in the primate. Pilot experiments in which AGRP was infused into the lateral ventricle (not reported here) were less successful, in that only two of five monkeys responded with a decrease in LH highlighting the importance of a central infusion site in the proximity of the arcuate nucleus of the hypothalamus. Even though pulsatile LH release was inhibited by infusion of AGRP into the third ventricle in all five tested monkeys, the response profile was different: the LH decrease was rapid in three animals but slower in the others, presumably reflecting varying locations of the catheter tip in relation to the arcuate nucleus.
Our data on the inhibitory effects of AGRP on pulsatile LH release are concordant with results obtained after NPY infusions, as well as into the third ventricle, in OVX rhesus monkeys. Similarly to AGRP, NPY reduced LH pulse frequency and mean LH levels, also indicating an inhibition of pulsatile GnRH-LH release (14). Taken together, the data from both experiments provide convincing support for a role of these two orexigenic neuropeptides in the control of the GnRH pulse generator. Significantly, AGRP and NPY are synthesized within the same neurons of the arcuate nucleus (11), which in the primate is also the presumed site of the GnRH pulse generator (20). Expression of both neuropeptides is increased with negative energy balance (11, 12, 13), a situation known to suppress the reproductive axis (1, 2, 3). The mechanisms by which NPY and AGRP influence energy homeostasis are, however, different. NPY acts through the activation of Y1 and Y5 receptors (9, 21, 22), whereas AGRP is an endogenous antagonist of MSH at MC3-R and MC4-R (6). Whether similar pathways mediate the effect of these two neuropeptides on the GnRH pulse generator remains to be fully investigated. However, suppressive effects of NPY on LH secretion in the rodent have been shown to be mediated by NPY Y1 and Y5 receptors (23, 24, 25). The mediatory role of MC3-R and/or MC4-R in the inhibitory effect of AGRP on pulsatile LH release is not known. Of interest are studies in the rodent that suggest that effects of a single central injection of AGRP on food intake are very potent (26, 27, 28), whereas NPY, even at very high dose, has little or no effect on food intake beyond 24 h (29). These differential effects of AGRP and NPY on food intake may reflect their selective pathways.
The observation that LH pulse frequency is decreased after AGRP administration suggests that this inhibitory effect of AGRP on the hypothalamic-pituitary-gonadal axis is mediated centrally through suppression of the GnRH pulse generator. Several pathways may be postulated to mediate this effect of AGRP. First, morphological data show that NPY/AGRP neurons in the arcuate nucleus have direct axonal contacts on the somata and proximal dendrites of GnRH neurons (30), suggesting that there may be a direct inhibitory effect of AGRP on the GnRH neurons. However, the type and location of receptors involved in this synaptic interaction are currently unknown.
Second, AGRP may suppress GnRH pulsatile release by enhancing the inhibitory effects of ?-endorphin (?-EP) on the GnRH neuron. ?-EP is derived from proopiomelanocortin, the same precursor that yields MSH, and proopiomelanocortin neurons are also known to contact GnRH neurons in the rodent (31). ?-EP is a strong inhibitor of the GnRH pulse generator, and we previously demonstrated that MSH can counteract the inhibitory effect of ?-EP on pulsatile LH release, suggesting that the ?-EP to MSH ratio is important in the control of pulsatile GnRH release (32). These data suggest that, because AGRP is an endogenous melanocortin receptor antagonist (6), elevations in AGRP levels may suppress pulsatile GnRH release by antagonizing endogenous MSH activity, thereby enhancing the inhibitory action of ?-EP.
Third, AGRP may suppress the GnRH pulse generator through activation of hypothalamus-pituitary-adrenal (HPA) pathways involved in the gonadotropin response to stressors because our present data also show that third-ventricle AGRP infusion in the OVX monkey increases cortisol release, confirming previous results (17). NPY has also been shown to stimulate the HPA endocrine axis in the intact male rhesus monkey and the dog (9, 33). Arcuate NPY/AGRP neurons project to the paraventricular nucleus, the location of CRH/AVP-containing neurons (34, 35). AGRP has been shown to increase the release of CRH and AVP, the two major stress-related neuropeptides, from hypothalamic explants (36). These HPA neuropeptides play a primary role in the control of pulsatile LH release in stress (37): administration of CRH or AVP results in a rapid decline in pulsatile LH release (18, 38), whereas inactivation of endogenous CRH or AVP activity by antagonists prevents the inhibition of pulsatile LH decrease that follows stressors (39, 40).
Fourth, AGRP may also directly activate opioid pathways and thereby inhibit pulsatile LH release. Studies have shown that the acute stimulatory effect of NPY and AGRP on food intake is mediated by μ- and -opiate receptors because administration of the nonspecific opioid receptor antagonist naloxone blocks this effect (41, 42). In the arcuate nucleus, NPY/AGRP neurons are synaptically linked to ?-EP-containing cells (43). These four putative pathways whereby AGRP may modulate pulsatile LH release remain to be fully investigated and may not be mutually exclusive.
Our experiments with AGRP showing an inhibition of pulsatile LH release, as those with NPY (14), were performed in the OVX monkey. In contrast, in intact male rats, intracerebroventricular administration of AGRP was shown to acutely increase basic pulsatile LH secretion (16). This difference in response may be species related or reflect the steroid environment. Indeed, effects of NPY on gonadotropin release are markedly influenced by the steroid status. For instance, whereas NPY inhibits pulsatile LH release in the castrated rat, sheep, rabbit, and monkey (14, 15, 44, 45), this peptide stimulates LH release in the intact male rat (46) and steroid-pretreated OVX rat (47). In the OVX-replaced monkey, however, estradiol appears to desensitize the animal to the inhibitory effect of NPY (14). Whether and how the steroid environment influences the LH response to AGRP remains to be investigated. It should also be mentioned that the mode of administration and/or site of AGRP or NPY infusion may also influence the LH response. It has indeed been shown that a pulsatile NPY infusion into the stalk-median eminence area is stimulatory to pulsatile GnRH release in the OVX monkey (48).
In summary, we have demonstrated that infusion of AGRP into the third ventricle suppresses pulsatile LH release in the OVX rhesus monkey, a result identical with that reported for NPY. We postulate that both AGRP and NPY may mediate the inhibitory effects of ghrelin and/or negative energy balance on the GnRH pulse generator and the reproductive system. The result of our study is relevant to physiopathological situations in which negative energy balance is combined with reproductive dysfunction because both AGRP and NPY mRNAs are up-regulated in food-deprived animals (11). Ghrelin, a powerful orexigenic peptide secreted by the stomach (49, 50, 51), is also chronically elevated in anorexia nervosa patients (52). Interestingly, in the rodent the effect of ghrelin on food intake is mediated by the NPY/AGRP neurons (53). We also recently demonstrated that a peripheral infusion of ghrelin in the OVX rhesus monkey decreases LH pulse frequency, indicating that ghrelin can also decrease the activity of the GnRH pulse generator (54). Further studies will be necessary to determine whether the inhibitory effect of ghrelin on the GnRH pulse generator is also mediated by NPY and/or AGRP.
Acknowledgments
We thank Dr. D. Van Vugt for his technological advice in regard to the third ventricular cannulation. Reagents for the monkey LH RIA were obtained with the help of Dr. A. F. Parlow (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA).
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