Y2 Receptor-Selective Agonist Delays the Estrogen-Induced Luteinizing Hormone Surge in Ovariectomized Ewes, but Y1-Receptor-Selective Agonis
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内分泌学杂志 2005年第2期
Prince Henry’s Institute of Medical Research (I.J.C., K.B.), Clayton, Melbourne, Victoria 3168, Australia; and Department of Physiology (K.B., A.J.T.), Monash University, Melbourne, Victoria 3800, Australia
Address all correspondence and requests for reprints to: Professor Iain J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: iain.clarke@phimr.monash.edu.au.
Abstract
Neuropeptide Y (NPY) plays a major role in the regulation of food intake, regulation of homeostasis, and neuroendocrine function. We have previously shown that third ventricular infusion of this peptide delays the estradiol benzoate-induced surge in LH secretion in ovariectomized ewes. To determine the receptor subtype that transmits this effect, we have now used the same model to infuse a Y1 receptor agonist [NPY Leu31 Pro34], a Y2 receptor agonist (PYY3–36), and a Y4 receptor agonist (pancreatic polypeptide). We monitored the surges in animals given these agonists or artificial cerebrospinal fluid by measuring plasma LH levels, and we also measured daily voluntary food intake (VFI). A low (7 μg/h) dose of Y2 agonist delayed the surge but did not affect VFI, whereas a higher dose (14 μg/h) stimulated VFI. A dose of 18 μg/h of the Y1 agonist did not affect surge generation but also stimulated VFI. A dose of 24 μg/h of Y4 agonist affected neither surge generation nor VFI. These specificities are different from those reported for the rat and human (in which a Y2 agonist causes reduction in VFI). We conclude that, in sheep, the negative regulation of the reproductive axis by NPY and Y-receptor agonists is effected via the Y2 receptors, whereas the orexigenic effects are most likely effected via the Y1 receptors.
Introduction
NEUROPEPTIDE Y (NPY) HAS been widely implicated in the regulation of food intake, metabolic balance, and neuroendocrine function. A high concentration of NPY-producing cells is found in the arcuate nucleus (ARC), with projections to the medial preoptic area (POA), anterior hypothalamic area, paraventricular nucleus, posterior hypothalamus, and the amygdaloid nucleus in the rat brain (1). A similar distribution of the peptide is seen in the ovine brain (2). NPY-containing terminals in the POA and median eminence come in close proximity with GnRH neurons in both the rat (3) and sheep (4), with one study demonstrating synaptic contacts between GnRH cell bodies and NPY in these areas in the rat (5). Li et al. (6) reported that NPY-producing cells of the ARC project to GnRH cell bodies and further showed presence of Y1 receptors in GnRH-immunopositive axons in the organum vasculosum of the lamina terminalis and median eminence but not on GnRH cell bodies. Because NPY is also produced in noradrenergic cells of the brain stem (7), observed NPY input to GnRH cells (8) is presumably due, in part, to presence of the peptide in noradrenergic afferents. Thus, there is ample anatomical evidence for regulation of GnRH neurons by NPY.
NPY may either inhibit or stimulate LH release in female rats, depending on the steroid milieu. LH secretion is suppressed in ovariectomized (OVX) animals (9, 10) but stimulated in intact or steroid-primed OVX rats (9). Similar results have been obtained in other species including the rabbit (11) and monkey (12). In OVX sheep (13, 14, 15) and cows (16), an inhibitory effect on LH secretion is also seen, but stimulatory effects observed in OVX-steroid-treated rats are not seen in the sheep (13). On the contrary, OVX steroid-treated ewes show a decrease in LH secretion when treated with NPY (13). Chronic treatment of rats with NPY suppressed reproductive function (17) and delayed sexual maturation (18), and more recent data suggest that this chronic effect is transmitted via the Y5 receptor (19). Central administration of NPY to estrogen-treated OVX ewes delayed or completely blocked the estrogen-induced LH surge (20). Furthermore, analysis of expression of NPY mRNA by in situ hybridization in cyclic ewes indicated that levels were highest during the luteal phase of the estrous cycle (20), a period when gonadotropin secretion is minimal. These data suggest that chronic elevation of brain NPY levels suppresses reproductive function.
To date, six receptor subtypes (Y1-Y6) have been found to mediate the actions of NPY and other endogenous ligands (21, 22, 23). In rats, the Y1 receptor transmits acute effects on the reproductive axis (24, 25, 26), but the Y4 and Y5 receptors also appear to be involved (19, 25). Further evidence of a role for the Y4 receptor in the regulation of reproductive function is provided in a study showing that the mating of Ob/Ob mice with Y4–/– mice rescued fertility in the former, although the crosses also affected metabolic parameters and reduced body weight (27). On the other hand, we found that central administration of a Y2 agonist suppressed LH secretion in OVX ewes, implicating this receptor subtype in the regulation of GnRH secretion in this species (15).
It is generally accepted that NPY stimulates food intake and encourages weight gain in a variety of species, such as rats (28) and sheep (29). These effects are thought to be effected via the Y1 and Y5 receptor subtypes in rodents (21, 30, 31). Further substantiation of the role of NPY in feeding behavior is demonstrated by up-regulation of NPY in the ARC of sheep with increased hunger drive due to food restriction (32) and down-regulation with leptin treatment that reduces voluntary food intake (33). Constantly elevated levels of NPY in the ARC of lean animals (34) could be the reason for hypogonadotropism in this state (35, 36). Recently it was shown that a selective Y2 receptor ligand [peptideYY3–36 (PYY)] could stimulate food intake in rodents and man (37) by presynaptic action of the peptide on NPY neurons within the brain, reducing hunger drive. Effects of this Y2 ligand on reproductive function were not determined in these studies.
The receptor subtypes through which NPY acts to regulate reproduction and appetite is not as well understood in species other than rodents. The aim of the present study was, therefore, to investigate which Y receptor subtypes might regulate these functions in the ewe. Because we previously showed that an infusion of NPY could delay/block the estrogen-induced LH surge in the OVX ewe, we further investigated the subtype of receptor that mediates this effect. We used a model of estrogen treatment of OVX ewes and infused a Y1 receptor agonist [NPY Leu31 Pro34], a Y2 receptor agonist (PYY3–36), and a Y4 receptor agonist [pancreatic polypeptide (PP)] into the third ventricle. Effects on the estrogen-induced surge were monitored by measuring plasma LH levels. Relative affinities of these ligands for the respective receptor subtypes have been previously detailed (21, 22). It was hypothesized that the Y2 agonist would selectively delay/block the LH surge, based on earlier results (15). In addition, we monitored voluntary food intake around the time of agonist infusion to determine which receptor subtype would mediate effects on feeding behavior.
Materials and Methods
The care and experimental use of the animals used in these experiments, was conducted in accordance with the requirements of the Australian Prevention of Cruelty to Animals Act 1986 and the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes. The Animal Ethics Committee of the Victorian Institute of Animal Science approved all the procedures used in this experiment.
Animals
The experiments used Corriedale ewes weighing between 50 and 60 kg and were conducted at Prince Henry’s Institute of Medical Research, Animal Research facility (Werribee, Victoria, Australia; 38 degrees latitude). During experimentation the animals were housed in individual pens and fed lucerne chaff and water ad libitum. For the duration of blood sampling, the animals were kept under continuous light. Before infusion and sampling, the animals were conditioned to pen housing and handling.
Surgical preparations cannulation and infusions
Ovariectomy and placement of guide tubes into the third cerebral ventricle (IIIV) were performed at least 6 wk before the commencement of the experiment. These procedures have been described previously (36). On the day before experimentation, each sheep received an indwelling jugular venous cannula, which was kept patent with heparinized (50 U/ml) saline. This was extended to the side of the pen with polyethylene tubing and closed with a three-way stopcock. Two hours before experimentation, custom-made stainless steel infusion cannulae were inserted into the IIIV and connected to polyethylene tubing that was connected to a syringe containing infusate in a battery-operated infusion pump (MS 16A infusion pump, Graseby Medical Ltd., Gold Coast, Australia). The pumps were secured to the side of the pen in a plastic case. The infusion lines and cannulae were primed with the appropriate infusate before insertion into the IIIV. The infusion rate was 62 μl/h.
Experimental design
All animals were injected (im) with 50 μg estradiol benzoate (EB; Intervet, New South Wales, Australia) in 1 ml peanut oil. This induces a time-delayed surge in LH secretion approximately 13–19 h in OVX ewes (15, 38). The study was a cross-over design in which ewes (n = 4) received either vehicle or Y-receptor agonist in alternate weeks (Fig. 1). Each agonist was administered to a separate group of animals.
FIG. 1. Experimental design. The hatched bars indicate periods of 0.5-h blood sampling.
Jugular venous blood samples (5 ml) were taken every 0.5 h from 1400 to 1600 h, and the animals were injected with EB at 1800 h. Blood samples were then taken between 0400 and 2400 h on the following day (10–30 h after EB injection). The EB-induced LH surge occurs between 12 and 20 h in this model. Samples were taken into heparinized tubes, centrifuged at 4 C, and stored at –20 C for LH assay.
Infusions of either artificial cerebrospinal fluid (aCSF; 150 mM NaCl, 1.2 mM CaCl2, 1 mM MgCl2, 2.8 mM KCl) commenced 1600 h (2 h before EB injection) and continued until 2200 h on the following day (28 h after EB). The agonist doses were as follows: 1) Y1 agonist human [Leu31 Pro34]NPY (Auspep, Melbourne, Australia), 18 μg/h; 2) Y2 agonist (human PYY3–36) (Auspep), 14 μg/h, followed by a repeat experiment using 7 μg/h; and 3) Y4 agonist (rat PP) (Auspep), 18 μg/h, followed by a repeat experiment using 24 μg/h.
Measurement of voluntary food intake (VFI)
The animals were offered 2–2.5 kg of lucerne chaff per day, depending on individual baseline VFI, and feeding was at 0900 h. VFI was calculated from refusals, commencing 1 wk before experimentation; data are presented for measurements taken over 4 d before EB injection and until 2 d after EB treatment.
RIA of LH
Concentrations of LH in plasma were determined in duplicate using the RIA previously described by Lee et al. (39). For 21 assays, assay sensitivity was 0.1–0.2 ng/ml, and the between-assay coefficients of variation were 13% at 3.0 ng/ml, 11% at 12.9 ng/ml, 8.8% at 5.8 ng/ml, and 10.1% at 22.6 ng/ml Within-assay coefficient of variation was less than 10% between 0.7 and 38.2 ng/ml (range).
Statistical analysis
Onset of the LH surge was defined as being when a rise in plasma LH concentrations occurred and was three times greater than the SD of the preceding LH concentration; the rise in plasma LH was sustained over at least 2 h; or peak levels during surge at least 10 times greater than levels immediately before surge onset. Mean time to surge onset was calculated for each treatment group, and the data were analyzed by repeated-measures ANOVA. Area under the curve (AUC) (LH vs. time) was calculated for the surge secretion of LH using SigmaPlot.
All data were analyzed by repeated-measures ANOVA after tests for homogeneity of variance. The within-subjects factor was treatment and the between-subjects factor was order of treatment. Least significant differences were used for post hoc testing of differences between means where appropriate.
Results
Effects of Y1, Y2, and Y4 agonists on the estrogen-induced LH surge
Vehicle (aCSF) treatment consistently caused a surge in LH secretion between 12 and 20 h after EB injection (Figs. 2 and 3). Infusion of 18 μg/h of the Y1 agonist did not alter the timing of onset. Infusion of 14 μg/h of the Y2 agonist blocked/delayed the surge, and a repeat experiment using 7 μg/h showed the same effect. Infusion of 18 μg/h of the Y4 agonist had no effect on the timing of the surge, and a repeat experiment at a higher dose (24 μg/h) showed the same result. Individual data for representative sheep given the Y1 agonist, the lower dose of the Y2 agonist, and the higher dose of the Y4 agonist are shown in Fig. 2, and mean data for time to onset of the LH surge are presented in Fig. 3.
FIG. 2. Examples of plasma LH profiles in OVX ewes that were given IIIV infusion of either artificial CSF or Y-receptor agonist. A, Data for an animal that received Leu31 Pro34 NPY (Y1 agonist) are shown. B, Data for an animal that received PYY3–36 (Y2 agonist) are shown. A shows data for an animal that received PP (Y4 agonist). Dotted lines show LH values during agonist treatment, and continuous lines show values during aCSF treatment.
FIG. 3. Mean (±SEM) time to the onset of the EB-induced surge in animals receiving IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist). Each agonist treatment was paired with aCSF treatment of the same animal.
AUC (plasma LH concentration vs. time) during the postinjection period was reduced by infusion of the Y2 agonist at both doses but was not affected by the infusion of the Y1 or Y4 agonist (Fig. 4).
FIG. 4. Mean (±SEM) AUC units (plasma LH concentration vs. time) 10–30 h after EB injection to OVX ewes with IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist). Each agonist treatment was paired with aCSF treatment of the same animal.
Effects of Y1, Y2, and Y4 agonists on VFI
Vehicle (aCSF) treatment consistently had no effect on VFI (Fig 5). Infusion of 18 μg/h of the Y1 agonist increased VFI by 36% (P < 0.02) over the 24 h after cessation of infusion. At the higher dose (14 μg/h), the Y2 agonist stimulated VFI (P < 0.05), but at the lower dose (7 μg/h), this effect was not seen. Neither dose of the Y4 agonist (18 and 24 μg/h) affected VFI.
FIG. 5. Mean (±SEM) VFI in animals receiving IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist) beginning at 1600 h on d 0 and continuing until 2200 h on d 1. The period of infusion is represented by the hatched bar. All animals received an im EB injection on d 0. Feeding and measurement of VFI was at 0900 h on each day, so that VFI shown as being recorded on d 2 relates to the 24-h period from 0900 h on the previous day. *, P < 0.05, **, P < 0.02 vs. aCSF.
Discussion
We previously showed that NPY infusion blocks or delays the EB-induced surge in LH secretion in OVX ewes (20), and the present study strongly suggests that this effect is transmitted via the Y2 receptor. Our earlier study tested the hypothesis that inhibitory effects of NPY would be mediated via the Y1 receptor by infusing a Y1 receptor antagonist, but no effects were seen (20). On the contrary, our present data strongly suggest that orexigenic effects of NPY or other Y ligands are most likely transmitted via the Y1 receptor, which is contrary to data from rats (40, 41, 42). Furthermore, contrary to data obtained in rats, we have not demonstrated a significant role for the Y4 receptor in either feeding behavior or the regulation of the reproductive system in the ewe. These data from a nonrodent species prompt the question as to whether PYY3–36 will have general utility as a means of reducing food intake.
We initially chose doses of peptide for infusion that were likely to be effective based on earlier work (20). A dose of 25 μg/h NPY blocks the EB-induced LH surge (20), but a dose of 10 μg/h does not (Clarke, I. J., unpublished data). With the Y1 agonist, an effect was seen on feeding behavior at a dose of 18 μg/h, so we did not increase or reduce this dose. At this dose, selectivity of effect was seen because there was no effect of [Leu31Pro34]NPY on the EB-induced LH surge. For the Y2 agonist, infusion of 14 μg/h affected both parameters, so we halved the dose; at the lower dose of 7 μg/h, selectivity was seen such that the EB-induced surge was blocked, but VFI was not affected. With the Y4 agonist, infusion of 18 μg/h had no effect on either VFI or the EB-induced LH surge, so we increased the dose to 24 μg/h; the result was the same. It is possible that higher doses of Y4 agonist would affect the generation of the LH surge and/or VFI, but within a range in which biological effects are clearly seen, it is likely that higher doses would cause side effects and/or the selectivity for particular subtypes of the Y receptor to be lost.
The Y1 agonist [Leu31Pro34]NPY has negligible affinity for Y2 but does have some affinity for the Y4 receptor and a 10-fold lower affinity for the Y5 receptor (21). Accordingly, high doses of this agonist could stimulate food intake through the Y5 receptor (21). The Y2 agonist that we used (PYY3–36) has negligible affinity for the Y1 or Y4 receptor but does appear to have some lower affinity for the Y5 receptor (21). The effect on VFI that was seen with the higher dose of the Y2 agonist could therefore have been due to action via the Y5 receptor. The Y4 agonist (PP) also has some Y5 receptor activity but less than the Y2 agonist (21); this agonist did not affect VFI or reproductive function. On balance, therefore, the high dose of Y2 agonist could have affected VFI via Y1 or Y5, and we cannot discount a possible role of Y5 in the VFI responses seen in Y1 agonist-treated animals. A much more extensive pharmacological experiment would be required to discriminate between Y1 and Y5 effects on VFI in sheep; [D-trp 32] is a selective Y5 agonist in vitro but has only weak short-term effects to stimulate VFI in rats (21).
Infusion of a Y1 antagonist in sheep produced no change to the onset of the estrogen-induced LH surge (20); neither did the Y1 agonist used in the present experiments. The dose of agonist used stimulated VFI, suggesting that the dose we used was biologically relevant. Both of these data sets strongly suggest that the Y1 receptor does not transmit effects of Y-receptor ligands on the reproductive axis, but this is contrary to the results obtained in rats. Thus, administration of a Y1 antagonist to rats compromised the steroid-induced LH surge (24, 26), suggesting that NPY action via the Y1 receptor is mandatory for the generation of the surge. Thus, a clear species difference exists between rats and sheep. The Y1 selective agonist that we used stimulated VFI, consistent with data from the rat. This agonist has some Y4 and Y5 activity in the rat (25), so the possibility exists that Y5 receptors may also be involved in feeding behavior in sheep.
Infusion of 14 μg/h of Y2 agonist PYY3–36 blocked/delayed the surge in all treated animals but also stimulated VFI. Accordingly, a further experiment was performed with half the dose, and we showed that this blocked the surge secretion of LH, without affecting VFI. This result is consistent with our previous work (15) showing that central infusion of a Y2 agonist reduced basal LH pulse amplitude in OVX ewes. Again, the data from the sheep are contrary to those obtained in the rat because a Y2 receptor agonist to rats produced no change to GnRH mRNA (43) or plasma LH concentrations (44). Whereas it has been shown that PYY3–36 reduces food intake in rats and humans (37, 45), we did not find a similar effect in sheep. Whether this Y2 ligand might affect the reproductive axis, or other endocrine functions, in other species including humans is not known, but the present study, albeit in sheep, suggests that wider investigation of the effects of treatment with this agent should perhaps be undertaken. Whereas PYY is a naturally occurring peptide in the blood (46, 47, 48) and fluctuations occur in relation to feeding, the effects of chronic elevation of the peptide by injection/infusion are not known.
Infusion of the Y4 agonist at two doses had no significant effect on either the estrogen-induced LH surge or VFI. These data are at variance with some reports of effects in other species. Whereas Raposinho et al. (25) found that the Y4 ligand (PP) did not suppress LH secretion in OVX rats, the data of Sainsbury et al. (27) suggest involvement of the Y4 receptor in the regulation of reproduction in mice. Our data are consistent with the results of the former study.
The Y2 agonist increased VFI at the higher dose used, and there was a possibility of nonspecificity of receptor action. Human PYY3–36 is relatively specific for Y2 but does have some low affinity for Y5 receptors (21). Mild side effects such as increased respiration rate and lethargy were also seen at the higher dose of the Y2 agonist, although we did not quantify this. NPY does have autonomic, cardiovascular, and respiratory effects (49, 50, 51), causing vasoconstriction (52) and modulating blood pressure (53) in rats. NPY also negatively regulates thermogenesis (54). Such effects might have accounted for the side effects seen. Because specificity of effect (on the EB-induced surge but not VFI) was seen at the lower dose, this is the most relevant dose to specify function via this receptor subtype.
Because NPY levels in the ARC are elevated and LH levels are reduced with reduction in body weight, the question arises as to whether the former causes the latter. Certainly data from chronic infusion studies suggest that this may be the case, but a causative link has not yet been established. On the other hand, elevated NPY levels in animals of low body weight may be more specifically related to feeding behavior. With reduced body weight, the response of the organism is to increase hunger drive, and this is effected via increased NPY expression. The factors that are responsible for the hypogonadotropic state in such conditions may include increased NPY expression, but other factors may also be involved. The present data are consistent with a role for NPY, and if infusion of a Y2 receptor antagonist restored LH secretion in animals of low body weight, this would provide strong evidence of such a link.
We found no effect of the selective Y4 agonist on feeding behavior in this study, strongly suggesting that this receptor subtype is not important in the regulation of feeding behavior in sheep, as in the rat (21). The Y4 receptor mRNA appears to be most abundant in the paraventricular nucleus and ARC (52, 55, 56) and may regulate other functions. This dose was thus increased to 24 μg and the experiment repeated. Again no significant change in the mean time to surge or VFI was observed. We did not administer a higher dose of PP because the Y4 receptor also mediates autonomic effects on the cardiovascular system (51).
The mechanism by which NPY or a Y2-specific agonist perturbs the positive feedback effect of estrogen to cause a GnRH/LH surge is not yet delineated, but various possibilities exist. GnRH cells in the ewe appear to receive synaptic input from NPY-producing elements, but as to whether these are noradrenergic projections that produce NPY or separate NPY-producing cells has not been delineated (8). Whereas the cells of the ARC to do not project directly to GnRH cell bodies, it is possible that NPY-producing cells of the POA do so (8, 57). In a recent study, Li et al. (6) found Y1 receptors on axons of GnRH cells but not the cell bodies, whereas there are no available data as to whether these cells produce Y2 receptors. The Y2 receptor is generally regarded as presynaptic, so it could modulate other inputs to GnRH cells. Alternatively, Y2 receptors could occur in the axonal projections of GnRH cells to the median eminence and/or at the level of the secretory terminals. Such possibilities remain to be investigated.
In summary, we have shown that a Y2-selective agonist selectively delays the EB-induced LH surge in OVX ewes but does not affect VFI. On the other hand, a Y1-selective agonist specifically stimulated VFI without affecting the EB-induced LH surge. A Y4-selective agonist affected neither the LH surge nor VFI.
Acknowledgments
We thank Bruce Doughton, Karen Briscoe, and Linda Morrish for assistance with the animal work; Alix Rao for assistance with assays; and Dr. A. F. Parlow (National Hormone and Peptide Program, NIDDK, Torrance, CA) for assay reagents.
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Address all correspondence and requests for reprints to: Professor Iain J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: iain.clarke@phimr.monash.edu.au.
Abstract
Neuropeptide Y (NPY) plays a major role in the regulation of food intake, regulation of homeostasis, and neuroendocrine function. We have previously shown that third ventricular infusion of this peptide delays the estradiol benzoate-induced surge in LH secretion in ovariectomized ewes. To determine the receptor subtype that transmits this effect, we have now used the same model to infuse a Y1 receptor agonist [NPY Leu31 Pro34], a Y2 receptor agonist (PYY3–36), and a Y4 receptor agonist (pancreatic polypeptide). We monitored the surges in animals given these agonists or artificial cerebrospinal fluid by measuring plasma LH levels, and we also measured daily voluntary food intake (VFI). A low (7 μg/h) dose of Y2 agonist delayed the surge but did not affect VFI, whereas a higher dose (14 μg/h) stimulated VFI. A dose of 18 μg/h of the Y1 agonist did not affect surge generation but also stimulated VFI. A dose of 24 μg/h of Y4 agonist affected neither surge generation nor VFI. These specificities are different from those reported for the rat and human (in which a Y2 agonist causes reduction in VFI). We conclude that, in sheep, the negative regulation of the reproductive axis by NPY and Y-receptor agonists is effected via the Y2 receptors, whereas the orexigenic effects are most likely effected via the Y1 receptors.
Introduction
NEUROPEPTIDE Y (NPY) HAS been widely implicated in the regulation of food intake, metabolic balance, and neuroendocrine function. A high concentration of NPY-producing cells is found in the arcuate nucleus (ARC), with projections to the medial preoptic area (POA), anterior hypothalamic area, paraventricular nucleus, posterior hypothalamus, and the amygdaloid nucleus in the rat brain (1). A similar distribution of the peptide is seen in the ovine brain (2). NPY-containing terminals in the POA and median eminence come in close proximity with GnRH neurons in both the rat (3) and sheep (4), with one study demonstrating synaptic contacts between GnRH cell bodies and NPY in these areas in the rat (5). Li et al. (6) reported that NPY-producing cells of the ARC project to GnRH cell bodies and further showed presence of Y1 receptors in GnRH-immunopositive axons in the organum vasculosum of the lamina terminalis and median eminence but not on GnRH cell bodies. Because NPY is also produced in noradrenergic cells of the brain stem (7), observed NPY input to GnRH cells (8) is presumably due, in part, to presence of the peptide in noradrenergic afferents. Thus, there is ample anatomical evidence for regulation of GnRH neurons by NPY.
NPY may either inhibit or stimulate LH release in female rats, depending on the steroid milieu. LH secretion is suppressed in ovariectomized (OVX) animals (9, 10) but stimulated in intact or steroid-primed OVX rats (9). Similar results have been obtained in other species including the rabbit (11) and monkey (12). In OVX sheep (13, 14, 15) and cows (16), an inhibitory effect on LH secretion is also seen, but stimulatory effects observed in OVX-steroid-treated rats are not seen in the sheep (13). On the contrary, OVX steroid-treated ewes show a decrease in LH secretion when treated with NPY (13). Chronic treatment of rats with NPY suppressed reproductive function (17) and delayed sexual maturation (18), and more recent data suggest that this chronic effect is transmitted via the Y5 receptor (19). Central administration of NPY to estrogen-treated OVX ewes delayed or completely blocked the estrogen-induced LH surge (20). Furthermore, analysis of expression of NPY mRNA by in situ hybridization in cyclic ewes indicated that levels were highest during the luteal phase of the estrous cycle (20), a period when gonadotropin secretion is minimal. These data suggest that chronic elevation of brain NPY levels suppresses reproductive function.
To date, six receptor subtypes (Y1-Y6) have been found to mediate the actions of NPY and other endogenous ligands (21, 22, 23). In rats, the Y1 receptor transmits acute effects on the reproductive axis (24, 25, 26), but the Y4 and Y5 receptors also appear to be involved (19, 25). Further evidence of a role for the Y4 receptor in the regulation of reproductive function is provided in a study showing that the mating of Ob/Ob mice with Y4–/– mice rescued fertility in the former, although the crosses also affected metabolic parameters and reduced body weight (27). On the other hand, we found that central administration of a Y2 agonist suppressed LH secretion in OVX ewes, implicating this receptor subtype in the regulation of GnRH secretion in this species (15).
It is generally accepted that NPY stimulates food intake and encourages weight gain in a variety of species, such as rats (28) and sheep (29). These effects are thought to be effected via the Y1 and Y5 receptor subtypes in rodents (21, 30, 31). Further substantiation of the role of NPY in feeding behavior is demonstrated by up-regulation of NPY in the ARC of sheep with increased hunger drive due to food restriction (32) and down-regulation with leptin treatment that reduces voluntary food intake (33). Constantly elevated levels of NPY in the ARC of lean animals (34) could be the reason for hypogonadotropism in this state (35, 36). Recently it was shown that a selective Y2 receptor ligand [peptideYY3–36 (PYY)] could stimulate food intake in rodents and man (37) by presynaptic action of the peptide on NPY neurons within the brain, reducing hunger drive. Effects of this Y2 ligand on reproductive function were not determined in these studies.
The receptor subtypes through which NPY acts to regulate reproduction and appetite is not as well understood in species other than rodents. The aim of the present study was, therefore, to investigate which Y receptor subtypes might regulate these functions in the ewe. Because we previously showed that an infusion of NPY could delay/block the estrogen-induced LH surge in the OVX ewe, we further investigated the subtype of receptor that mediates this effect. We used a model of estrogen treatment of OVX ewes and infused a Y1 receptor agonist [NPY Leu31 Pro34], a Y2 receptor agonist (PYY3–36), and a Y4 receptor agonist [pancreatic polypeptide (PP)] into the third ventricle. Effects on the estrogen-induced surge were monitored by measuring plasma LH levels. Relative affinities of these ligands for the respective receptor subtypes have been previously detailed (21, 22). It was hypothesized that the Y2 agonist would selectively delay/block the LH surge, based on earlier results (15). In addition, we monitored voluntary food intake around the time of agonist infusion to determine which receptor subtype would mediate effects on feeding behavior.
Materials and Methods
The care and experimental use of the animals used in these experiments, was conducted in accordance with the requirements of the Australian Prevention of Cruelty to Animals Act 1986 and the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes. The Animal Ethics Committee of the Victorian Institute of Animal Science approved all the procedures used in this experiment.
Animals
The experiments used Corriedale ewes weighing between 50 and 60 kg and were conducted at Prince Henry’s Institute of Medical Research, Animal Research facility (Werribee, Victoria, Australia; 38 degrees latitude). During experimentation the animals were housed in individual pens and fed lucerne chaff and water ad libitum. For the duration of blood sampling, the animals were kept under continuous light. Before infusion and sampling, the animals were conditioned to pen housing and handling.
Surgical preparations cannulation and infusions
Ovariectomy and placement of guide tubes into the third cerebral ventricle (IIIV) were performed at least 6 wk before the commencement of the experiment. These procedures have been described previously (36). On the day before experimentation, each sheep received an indwelling jugular venous cannula, which was kept patent with heparinized (50 U/ml) saline. This was extended to the side of the pen with polyethylene tubing and closed with a three-way stopcock. Two hours before experimentation, custom-made stainless steel infusion cannulae were inserted into the IIIV and connected to polyethylene tubing that was connected to a syringe containing infusate in a battery-operated infusion pump (MS 16A infusion pump, Graseby Medical Ltd., Gold Coast, Australia). The pumps were secured to the side of the pen in a plastic case. The infusion lines and cannulae were primed with the appropriate infusate before insertion into the IIIV. The infusion rate was 62 μl/h.
Experimental design
All animals were injected (im) with 50 μg estradiol benzoate (EB; Intervet, New South Wales, Australia) in 1 ml peanut oil. This induces a time-delayed surge in LH secretion approximately 13–19 h in OVX ewes (15, 38). The study was a cross-over design in which ewes (n = 4) received either vehicle or Y-receptor agonist in alternate weeks (Fig. 1). Each agonist was administered to a separate group of animals.
FIG. 1. Experimental design. The hatched bars indicate periods of 0.5-h blood sampling.
Jugular venous blood samples (5 ml) were taken every 0.5 h from 1400 to 1600 h, and the animals were injected with EB at 1800 h. Blood samples were then taken between 0400 and 2400 h on the following day (10–30 h after EB injection). The EB-induced LH surge occurs between 12 and 20 h in this model. Samples were taken into heparinized tubes, centrifuged at 4 C, and stored at –20 C for LH assay.
Infusions of either artificial cerebrospinal fluid (aCSF; 150 mM NaCl, 1.2 mM CaCl2, 1 mM MgCl2, 2.8 mM KCl) commenced 1600 h (2 h before EB injection) and continued until 2200 h on the following day (28 h after EB). The agonist doses were as follows: 1) Y1 agonist human [Leu31 Pro34]NPY (Auspep, Melbourne, Australia), 18 μg/h; 2) Y2 agonist (human PYY3–36) (Auspep), 14 μg/h, followed by a repeat experiment using 7 μg/h; and 3) Y4 agonist (rat PP) (Auspep), 18 μg/h, followed by a repeat experiment using 24 μg/h.
Measurement of voluntary food intake (VFI)
The animals were offered 2–2.5 kg of lucerne chaff per day, depending on individual baseline VFI, and feeding was at 0900 h. VFI was calculated from refusals, commencing 1 wk before experimentation; data are presented for measurements taken over 4 d before EB injection and until 2 d after EB treatment.
RIA of LH
Concentrations of LH in plasma were determined in duplicate using the RIA previously described by Lee et al. (39). For 21 assays, assay sensitivity was 0.1–0.2 ng/ml, and the between-assay coefficients of variation were 13% at 3.0 ng/ml, 11% at 12.9 ng/ml, 8.8% at 5.8 ng/ml, and 10.1% at 22.6 ng/ml Within-assay coefficient of variation was less than 10% between 0.7 and 38.2 ng/ml (range).
Statistical analysis
Onset of the LH surge was defined as being when a rise in plasma LH concentrations occurred and was three times greater than the SD of the preceding LH concentration; the rise in plasma LH was sustained over at least 2 h; or peak levels during surge at least 10 times greater than levels immediately before surge onset. Mean time to surge onset was calculated for each treatment group, and the data were analyzed by repeated-measures ANOVA. Area under the curve (AUC) (LH vs. time) was calculated for the surge secretion of LH using SigmaPlot.
All data were analyzed by repeated-measures ANOVA after tests for homogeneity of variance. The within-subjects factor was treatment and the between-subjects factor was order of treatment. Least significant differences were used for post hoc testing of differences between means where appropriate.
Results
Effects of Y1, Y2, and Y4 agonists on the estrogen-induced LH surge
Vehicle (aCSF) treatment consistently caused a surge in LH secretion between 12 and 20 h after EB injection (Figs. 2 and 3). Infusion of 18 μg/h of the Y1 agonist did not alter the timing of onset. Infusion of 14 μg/h of the Y2 agonist blocked/delayed the surge, and a repeat experiment using 7 μg/h showed the same effect. Infusion of 18 μg/h of the Y4 agonist had no effect on the timing of the surge, and a repeat experiment at a higher dose (24 μg/h) showed the same result. Individual data for representative sheep given the Y1 agonist, the lower dose of the Y2 agonist, and the higher dose of the Y4 agonist are shown in Fig. 2, and mean data for time to onset of the LH surge are presented in Fig. 3.
FIG. 2. Examples of plasma LH profiles in OVX ewes that were given IIIV infusion of either artificial CSF or Y-receptor agonist. A, Data for an animal that received Leu31 Pro34 NPY (Y1 agonist) are shown. B, Data for an animal that received PYY3–36 (Y2 agonist) are shown. A shows data for an animal that received PP (Y4 agonist). Dotted lines show LH values during agonist treatment, and continuous lines show values during aCSF treatment.
FIG. 3. Mean (±SEM) time to the onset of the EB-induced surge in animals receiving IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist). Each agonist treatment was paired with aCSF treatment of the same animal.
AUC (plasma LH concentration vs. time) during the postinjection period was reduced by infusion of the Y2 agonist at both doses but was not affected by the infusion of the Y1 or Y4 agonist (Fig. 4).
FIG. 4. Mean (±SEM) AUC units (plasma LH concentration vs. time) 10–30 h after EB injection to OVX ewes with IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist). Each agonist treatment was paired with aCSF treatment of the same animal.
Effects of Y1, Y2, and Y4 agonists on VFI
Vehicle (aCSF) treatment consistently had no effect on VFI (Fig 5). Infusion of 18 μg/h of the Y1 agonist increased VFI by 36% (P < 0.02) over the 24 h after cessation of infusion. At the higher dose (14 μg/h), the Y2 agonist stimulated VFI (P < 0.05), but at the lower dose (7 μg/h), this effect was not seen. Neither dose of the Y4 agonist (18 and 24 μg/h) affected VFI.
FIG. 5. Mean (±SEM) VFI in animals receiving IIIV infusions of Leu31 Pro34 NPY (Y1 agonist), PYY3–36 (Y2 agonist), or PP (Y4 agonist) beginning at 1600 h on d 0 and continuing until 2200 h on d 1. The period of infusion is represented by the hatched bar. All animals received an im EB injection on d 0. Feeding and measurement of VFI was at 0900 h on each day, so that VFI shown as being recorded on d 2 relates to the 24-h period from 0900 h on the previous day. *, P < 0.05, **, P < 0.02 vs. aCSF.
Discussion
We previously showed that NPY infusion blocks or delays the EB-induced surge in LH secretion in OVX ewes (20), and the present study strongly suggests that this effect is transmitted via the Y2 receptor. Our earlier study tested the hypothesis that inhibitory effects of NPY would be mediated via the Y1 receptor by infusing a Y1 receptor antagonist, but no effects were seen (20). On the contrary, our present data strongly suggest that orexigenic effects of NPY or other Y ligands are most likely transmitted via the Y1 receptor, which is contrary to data from rats (40, 41, 42). Furthermore, contrary to data obtained in rats, we have not demonstrated a significant role for the Y4 receptor in either feeding behavior or the regulation of the reproductive system in the ewe. These data from a nonrodent species prompt the question as to whether PYY3–36 will have general utility as a means of reducing food intake.
We initially chose doses of peptide for infusion that were likely to be effective based on earlier work (20). A dose of 25 μg/h NPY blocks the EB-induced LH surge (20), but a dose of 10 μg/h does not (Clarke, I. J., unpublished data). With the Y1 agonist, an effect was seen on feeding behavior at a dose of 18 μg/h, so we did not increase or reduce this dose. At this dose, selectivity of effect was seen because there was no effect of [Leu31Pro34]NPY on the EB-induced LH surge. For the Y2 agonist, infusion of 14 μg/h affected both parameters, so we halved the dose; at the lower dose of 7 μg/h, selectivity was seen such that the EB-induced surge was blocked, but VFI was not affected. With the Y4 agonist, infusion of 18 μg/h had no effect on either VFI or the EB-induced LH surge, so we increased the dose to 24 μg/h; the result was the same. It is possible that higher doses of Y4 agonist would affect the generation of the LH surge and/or VFI, but within a range in which biological effects are clearly seen, it is likely that higher doses would cause side effects and/or the selectivity for particular subtypes of the Y receptor to be lost.
The Y1 agonist [Leu31Pro34]NPY has negligible affinity for Y2 but does have some affinity for the Y4 receptor and a 10-fold lower affinity for the Y5 receptor (21). Accordingly, high doses of this agonist could stimulate food intake through the Y5 receptor (21). The Y2 agonist that we used (PYY3–36) has negligible affinity for the Y1 or Y4 receptor but does appear to have some lower affinity for the Y5 receptor (21). The effect on VFI that was seen with the higher dose of the Y2 agonist could therefore have been due to action via the Y5 receptor. The Y4 agonist (PP) also has some Y5 receptor activity but less than the Y2 agonist (21); this agonist did not affect VFI or reproductive function. On balance, therefore, the high dose of Y2 agonist could have affected VFI via Y1 or Y5, and we cannot discount a possible role of Y5 in the VFI responses seen in Y1 agonist-treated animals. A much more extensive pharmacological experiment would be required to discriminate between Y1 and Y5 effects on VFI in sheep; [D-trp 32] is a selective Y5 agonist in vitro but has only weak short-term effects to stimulate VFI in rats (21).
Infusion of a Y1 antagonist in sheep produced no change to the onset of the estrogen-induced LH surge (20); neither did the Y1 agonist used in the present experiments. The dose of agonist used stimulated VFI, suggesting that the dose we used was biologically relevant. Both of these data sets strongly suggest that the Y1 receptor does not transmit effects of Y-receptor ligands on the reproductive axis, but this is contrary to the results obtained in rats. Thus, administration of a Y1 antagonist to rats compromised the steroid-induced LH surge (24, 26), suggesting that NPY action via the Y1 receptor is mandatory for the generation of the surge. Thus, a clear species difference exists between rats and sheep. The Y1 selective agonist that we used stimulated VFI, consistent with data from the rat. This agonist has some Y4 and Y5 activity in the rat (25), so the possibility exists that Y5 receptors may also be involved in feeding behavior in sheep.
Infusion of 14 μg/h of Y2 agonist PYY3–36 blocked/delayed the surge in all treated animals but also stimulated VFI. Accordingly, a further experiment was performed with half the dose, and we showed that this blocked the surge secretion of LH, without affecting VFI. This result is consistent with our previous work (15) showing that central infusion of a Y2 agonist reduced basal LH pulse amplitude in OVX ewes. Again, the data from the sheep are contrary to those obtained in the rat because a Y2 receptor agonist to rats produced no change to GnRH mRNA (43) or plasma LH concentrations (44). Whereas it has been shown that PYY3–36 reduces food intake in rats and humans (37, 45), we did not find a similar effect in sheep. Whether this Y2 ligand might affect the reproductive axis, or other endocrine functions, in other species including humans is not known, but the present study, albeit in sheep, suggests that wider investigation of the effects of treatment with this agent should perhaps be undertaken. Whereas PYY is a naturally occurring peptide in the blood (46, 47, 48) and fluctuations occur in relation to feeding, the effects of chronic elevation of the peptide by injection/infusion are not known.
Infusion of the Y4 agonist at two doses had no significant effect on either the estrogen-induced LH surge or VFI. These data are at variance with some reports of effects in other species. Whereas Raposinho et al. (25) found that the Y4 ligand (PP) did not suppress LH secretion in OVX rats, the data of Sainsbury et al. (27) suggest involvement of the Y4 receptor in the regulation of reproduction in mice. Our data are consistent with the results of the former study.
The Y2 agonist increased VFI at the higher dose used, and there was a possibility of nonspecificity of receptor action. Human PYY3–36 is relatively specific for Y2 but does have some low affinity for Y5 receptors (21). Mild side effects such as increased respiration rate and lethargy were also seen at the higher dose of the Y2 agonist, although we did not quantify this. NPY does have autonomic, cardiovascular, and respiratory effects (49, 50, 51), causing vasoconstriction (52) and modulating blood pressure (53) in rats. NPY also negatively regulates thermogenesis (54). Such effects might have accounted for the side effects seen. Because specificity of effect (on the EB-induced surge but not VFI) was seen at the lower dose, this is the most relevant dose to specify function via this receptor subtype.
Because NPY levels in the ARC are elevated and LH levels are reduced with reduction in body weight, the question arises as to whether the former causes the latter. Certainly data from chronic infusion studies suggest that this may be the case, but a causative link has not yet been established. On the other hand, elevated NPY levels in animals of low body weight may be more specifically related to feeding behavior. With reduced body weight, the response of the organism is to increase hunger drive, and this is effected via increased NPY expression. The factors that are responsible for the hypogonadotropic state in such conditions may include increased NPY expression, but other factors may also be involved. The present data are consistent with a role for NPY, and if infusion of a Y2 receptor antagonist restored LH secretion in animals of low body weight, this would provide strong evidence of such a link.
We found no effect of the selective Y4 agonist on feeding behavior in this study, strongly suggesting that this receptor subtype is not important in the regulation of feeding behavior in sheep, as in the rat (21). The Y4 receptor mRNA appears to be most abundant in the paraventricular nucleus and ARC (52, 55, 56) and may regulate other functions. This dose was thus increased to 24 μg and the experiment repeated. Again no significant change in the mean time to surge or VFI was observed. We did not administer a higher dose of PP because the Y4 receptor also mediates autonomic effects on the cardiovascular system (51).
The mechanism by which NPY or a Y2-specific agonist perturbs the positive feedback effect of estrogen to cause a GnRH/LH surge is not yet delineated, but various possibilities exist. GnRH cells in the ewe appear to receive synaptic input from NPY-producing elements, but as to whether these are noradrenergic projections that produce NPY or separate NPY-producing cells has not been delineated (8). Whereas the cells of the ARC to do not project directly to GnRH cell bodies, it is possible that NPY-producing cells of the POA do so (8, 57). In a recent study, Li et al. (6) found Y1 receptors on axons of GnRH cells but not the cell bodies, whereas there are no available data as to whether these cells produce Y2 receptors. The Y2 receptor is generally regarded as presynaptic, so it could modulate other inputs to GnRH cells. Alternatively, Y2 receptors could occur in the axonal projections of GnRH cells to the median eminence and/or at the level of the secretory terminals. Such possibilities remain to be investigated.
In summary, we have shown that a Y2-selective agonist selectively delays the EB-induced LH surge in OVX ewes but does not affect VFI. On the other hand, a Y1-selective agonist specifically stimulated VFI without affecting the EB-induced LH surge. A Y4-selective agonist affected neither the LH surge nor VFI.
Acknowledgments
We thank Bruce Doughton, Karen Briscoe, and Linda Morrish for assistance with the animal work; Alix Rao for assistance with assays; and Dr. A. F. Parlow (National Hormone and Peptide Program, NIDDK, Torrance, CA) for assay reagents.
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