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Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions
BBRC Biochemical and Biophysical Research Communications 311 (2003) 721–727 www.elsevier.com/locate/ybbrc
Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions Hong Quan, Toshiya Funabashi,* Miyako Furuta, and Fukuko Kimura Department of Neuroendocrinology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan Received 1 October 2003
Abstract We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 ll) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Neuromedin U; Intracerebroventricular injection; Pulsatile LH secretion; Ovariectomized rats; Fasting; Feeding condition; Food deprivation; Luteinizing hormone; Female rats
Neuromedin U (NMU), a 23-amino-acid peptide, is a brain–gut peptide originally isolated from the spinal cord in pigs and later in many other species [1,2]. NMU acts through two receptors named NMU1R and NMU2R. The expression of rat NMU1R is abundant in peripheral tissues, but is sparse in the brain [3–6]. The expression of rat NMU2R is, on the other hand, mostly restricted to specific regions in the brain, such as the paraventricular nucleus (PVN), along the wall of the third ventricle in the hypothalamus, and the hippocampus [3,7,8]. Although NMU was initially shown to play a role in smooth muscle contraction [1], the expression of NMU2R in the brain suggests that NMU also plays a physiological role in the brain. Indeed, NMU induces adrenocorticotropic hormone (ACTH) secretion [9], suggesting involvement in the control of stress responses. Recently, it was also reported that central administration of NMU suppresses food intake in rats [10–12]. In addition, NMU mRNA is expressed * Corresponding author. Fax: +81-45-787-2578. E-mail address: [email protected] (T. Funabashi).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.052
abundantly in the ventromedial hypothalamic area which is an important site for inducing satiety, and its level is reduced in rats fasted for 48 h [3]. Thus, NMU appears to act in the hypothalamus as a satiety factor. Many neuropeptides which influence feeding behavior also affect reproductive functions. For example, ghrelin and orexin, which induce food intake and act as orexigenic factors [13,14], suppress pulsatile luteinizing hormone (LH) secretion [15–17]. In contrast, leptin acts as a satiety factor [18–20] and reverses the inhibition of pulsatile LH secretion by fasting [21]. Glucagon-like peptide-1 also acts as a satiety factor [22], stimulating LH secretion but decreasing its own level in the hypothalamus of male rats fasted for 48 h, which indicates a possible association of the satiety factor with the low LH levels in hungry animals [23]. These reports indicate that a satiety factor can prevent fasting-induced suppression of the pulsatile LH secretion. Since NMU acts as a satiety factor [3,10,11], we wondered whether NMU restores the pulsatile LH secretion which would have been inhibited by fasting. We therefore examined whether central administration of NMU influenced the
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pulsatile LH secretion in normally feeding rats or in 48h fasted rats. We report here that, different from leptin, NMU suppresses the pulsatile LH secretion under the 48-h fasted condition.
Materials and methods Animals. Female rats of Wistar strain (Charles River, Yokohama, Japan) were obtained at 7 weeks of age and maintained under controlled lighting conditions (lights on 05:00–19:00 h) with food and water available ad libitum. They were ovariectomized (OVX) and, for the intracerebroventricular (icv) injection, stereotaxically implanted with a guide cannula into the third ventricle according to the atlas of Albe-Fessard et al. [24] (stereotaxic coordinates: A ¼ 6:0, V ¼ 2:0, and L ¼ 0:0) under sodium pentobarbital (31.5 mg/kg body weight) anesthesia as reported previously [25]. All animal housing and surgical procedures were carried out according to the Guidelines laid down by the Institutional Animal Care and Use Committee of the Yokohama City University, Graduate School of Medicine. Two to three weeks after the brain surgery, each rat was handled daily for 10 min 1 week before the blood sampling. They were divided into two experimental groups; in experiment 1, the rats had access to an unlimited supply of food (normal feeding condition) and in experiment 2, the rats were deprived of food for 48 h (48-h fasted condition). An intraatrial cannula was implanted through the jugular vein on the day before the blood sampling. The blood sampling, approximately 120 ll each, was performed under freely moving conditions at 6-min intervals from 09:00 to 12:00 h. An equal volume of heparinized saline (2 IU/ml) was injected after each sampling. After the first hour of blood sampling, rat NMU (Peptide Institute, Osaka, Japan) dissolved in artificial cerebrospinal fluid (aCSF, 1 nmol/3 ll) was injected into the third ventricle through the guide cannula using a Hamilton microsyringe. This dose of NMU has been shown to induce satiety in rats [10]. The same volume of aCSF was injected as a control. Further blood samplings were performed for the second and third hours. Thus, the rats were arranged in a total of four groups: OVX rats under the normal feeding condition injected with aCSF (aCSF-injected OVX rats, n ¼ 7) or NMU (NMU-injected OVX rats, n ¼ 7) for experiment 1; OVX rats under the 48-h fasted condition injected with aCSF (aCSF-injected OVX rats, n ¼ 6) or NMU (NMU-injected OVX rats, n ¼ 7) for experiment 2. In a preliminary experiment, we found that estrogen replacement at a dose which significantly potentiated the inhibitory effect of orexin on the pulsatile LH secretion [16] did not affect the inhibitory effect of NMU under the 48-h fasted condition. Therefore, we used OVX rats without estrogen replacement in the present study. Serum concentrations of LH were measured by double antibody radioimmunoassay with materials supplied by the NIDDK and generously donated by Dr. K. Wakabayashi (Gunma University, Maebashi, Japan). The reference standard for LH was NIDDK rat LH-RP-4. However, the amount of LH is expressed in terms of NIH LH-S1. LH was determined in a single assay for experiment 1 and experiment 2. The minimally detectable amount of LH (95% confi-
dence limits of buffer controls) was 0.12 ng/ml (experiment 1) and 0.27 ng/ml (experiment 2), and the intraassay coefficient of variation (CV) for LH was 7.6% (experiment 1) and 8.7% (experiment 2). Statistics. The LH pulse detection was based on methods reported previously [26,27]. The analysis of LH pulsatility included the determination of pulse frequency (number of LH pulses in each of the first, second, and third hours) and pulse amplitude (difference between the peak and the pre-peak nadir) in each of the first, second, and third hours. The pulse amplitude was averaged for each of the 3 h in each rat and then mean amplitude was calculated for each group. In addition, the interpulse interval was calculated before (mean interpulse interval for the first hour) and after the injection (interpulse interval between the first and second pulse after the injection). If a first pulse after the injection did not appear within 24 min, which is the average interpulse interval without treatment (the first hour, Table 1), the latest pulse before the injection was defined as the first pulse after the injection (for example, Fig. 1 #15 and Fig. 3 #12). Analysis of variance (ANOVA) followed by Fisher’s PLSD post hoc comparison was used to test the statistical significance and significance was accepted at p < 0:05. Group data of serum LH concentrations over a 3-h period were also obtained by averaging values at the corresponding time. The data obtained this way were analyzed by paired t test. To this end, LH concentration was averaged for the first hour in each rat and compared to the corresponding time in each treatment group.
Results Experiment 1 Fig. 1 shows representative individual LH profiles and the mean LH concentration before and after icv injection of aCSF or NMU in OVX rats under the normal feeding condition. The pulsatile fluctuation of serum LH was evident during the blood sampling in aCSF-injected OVX rats, suggesting that icv injection of aCSF had no significant effect on the pulsatile LH secretion. NMU injection, on the other hand, seemed to inhibit the pulsatile LH secretion, since the serum LH concentration at the corresponding time after the injection was significantly lower than before the injection (Fig. 1, lower column). Two-way ANOVA, however, showed no significant effects of the treatment (p > 0:5, aCSF and NMU) and the time (p > 0:1, the first, second, and third hours) without the interaction (p > 0:1) on the pulse parameters, such as the frequency and the amplitude (Fig. 2). Two-way ANOVA of the interpulse interval, on the other hand, showed significance for the effects of both the treatment (p < 0:05, aCSF and NMU) and the time (p < 0:001, before and after the injection) with a significant interaction (p < 0:05).
Table 1 Effects of icv injection of aCSF or NMU on the interpulse interval in OVX rats under the normal feeding or the 48-h fasted condition Treatment
Normal feeding condition N
Before
After
N
Before
After
aCSF NMU
7 7
19.2 1.7 23.2 4.2
19.7 1.7 36.0 2.3*
6 7
25.0 1.8 20.7 1.8
28.0 1.3 54.9 6.7**
48-h fasted condition
* and ** indicate p < 0:005 and p < 0:001 vs the value before the injection, respectively. See the text for further statistical details.
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Fig. 1. Representative examples of effects of icv injection of aCSF (left panels) or NMU (right panels) on the serum concentration of LH in OVX rats under the normal feeding condition. The arrows indicate the time of icv injection of NMU or aCSF. Triangles indicate statistically defined LH pulses. The mean ( SEM) of LH concentrations at the corresponding time is shown in the lower panels. * indicates p < 0:05 vs. the value of the first hour. See the text for further statistical details.
Since the interaction was significant, we analyzed the data by one-way ANOVA (p < 0:001) and found by post hoc comparison that, in NMU-injected OVX rats, the interpulse interval after the injection was significantly larger than before the injection (Table 1, p < 0:005). Further, the interpulse interval in NMUinjected rats after the injection was significantly larger than in aCSF-injected rats before (p < 0:001) and after the injection (p < 0:001). ANOVA of the pulse, frequency (Fig. 2) and the interpulse interval (Table 1), indicated that, under the normal feeding condition, the pulsatile LH secretion was not markedly influenced by NMU injection (Fig. 2), but there was a statistically significant suppression by NMU injection in the interpulse interval (Table 1).
Experiment 2 In OVX rats under the 48-h fasted condition, icv injection of aCSF did not affect the pulsatile LH secretion, as shown in Fig. 3, since the pulsatile fluctuation of LH appeared to be comparable to the one in aCSF-injected OVX rats under the normal feeding condition. This suggests that the fasting condition used in the present study had no significant effect on the pulsatile LH secretion in OVX rats. ANOVA within the first hour of aCSF-injected OVX rats showed that there was no significant difference in the frequency (p > 0:5) or the amplitude (p > 0:5) between the normal feeding condition and the 48-h fasted condition. NMU injection, on the other hand, appeared to quickly suppress the pulsatile
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was no significant difference in the pulsatility between aCSF- and NMU-injected OVX rats before injection (p > 0:1). As shown in Table 1, two-way ANOVA of the interpulse interval showed a significant effect of the treatment (aCSF or NMU, P < 0:01) and the time (before and after the injection, p < 0:0001) with a significant interaction (p < 0:001). In one-way ANOVA (p < 0:0001) followed by post hoc comparison, the interpulse interval in NMU-injected OVX rats after the injection was significantly larger than in NMU-injected OVX rats before the injection (p < 0:001), aCSF-injected OVX rats before the injection (p < 0:001), and aCSF-injected OVX rats after the injection (p < 0:001). Finally, two-way ANOVA of the amplitude (Fig. 4) showed no significant effects of the treatment (p > 0:5, aCSF and NMU) and the time (p > 0:1, the first, second, and third hours) without the interaction (p > 0:1).
Discussion
Fig. 2. Effects of icv injection of aCSF (n ¼ 7) or NMU (n ¼ 7) on the pulse frequency and the pulse amplitude in OVX rats under the normal feeding condition. They are expressed as the mean of the first hour (designated as 1 under the column), second hour (2), and third hour (3). Icv injections were made after the first hour. No significant difference was found in these parameters.
LH secretion, as shown in Fig. 3. The serum LH concentration at the corresponding time after NMU injection was significantly lower than before the injection (Fig. 3, lower column). Two-way ANOVA of the frequency (Fig. 4) did not show any significant effect of the treatment (aCSF or NMU, P > 0:1), but showed a significant effect of the time (the first, second, and third hours, p < 0:01) with a significant interaction (p < 0:05). Since the interaction was significant, we analyzed the data by one-way ANOVA (p < 0:01). Post hoc comparison showed that the frequency in the second hour of NMU-injected OVX rats was significantly smaller than in the first hour of NMU-injected OVX rats (p < 0:0001) and aCSF-injected OVX rats (p < 0:005). In NMU-injected rats, the frequency in the third hour was significantly smaller than in the first hour (p < 0:05). Compared to aCSF-injected OVX rats at the corresponding time, the frequency was significantly smaller in the second hour in NMU-injected OVX rats (p < 0:01), but not in the first hour, suggesting that there
The present study shows for the first time that the central administration of NMU inhibits the pulsatile LH secretion and this effect is significantly enhanced under the 48-h fasted condition, which by itself does not markedly affect the pulsatile LH secretion. Since satiety factors such as leptin and glucagon-like peptide-1 stimulate the LH secretion [21,23], while orexinergic factors such as ghrelin, orexin, and melanin-concentrating hormone inhibit it [15,16,28], the inhibitory action of NMU on the LH secretion shown in the present study is in contrast to the action of satiety factors. It is therefore appears that NMU plays a different role in the control of the pulsatile LH secretion than other satiety-inducing peptides. In rats, the GnRH pulse generator in the hypothalamus governs the pulsatile LH secretion [29] and the frequency of LH pulses in general reflects the frequency of GnRH pulses [30]. Therefore, the observation that the frequency and the interpulse interval, but not the amplitude, of LH pulses was altered by NMU injection suggests that NMU acted on the hypothalamus but not on the anterior pituitary. If this is the case, the next question is how NMU inhibits the pulsatile GnRH secretion. It has been reported that a central administration of NMU stimulates ACTH and corticosterone secretions in male rats [31] and that NMU increases the release of CRH from hypothalamic explants in vitro [32]. Since NMU activates neurons in the PVN [32,33], it is likely that NMU induces the activation of CRH neurons in the PVN and, at least in part, neuroendocrine responses to NMU are mediated by CRH neurons. In fact, NMU-induced activation of locomotor activity, face-washing behavior, and grooming were partially abolished by pretreatment with an antagonist of CRH, a-helical CRH, or anti-CRH IgG, and these effects of
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Fig. 3. Representative examples of effects of icv injection of aCSF (left panels) or NMU (right panels) on the serum concentration of LH in OVX rats under the 48-h fasted condition. The arrows indicate the time of icv injection of NMU or aCSF. Triangles indicate statistically defined LH pulses. The mean ( SEM) of LH concentrations at the corresponding time is shown in the lower panels. * indicates p < 0:05 vs. the value of the first hour. See the text for further statistical details.
NMU were not observed in CRH knockout mice [34]. On the other hand, the involvement of CRH in the control of LH secretion has been suggested. It has been reported that the icv injection of CRH inhibited the pulsatile LH secretion in a dose-related manner in OVX female rats [35] irrespective of the presence of estrogen [36], indicating that the CRH system plays an inhibitory role in the LH secretion. It has also been reported that CRH inhibits the pulsatile LH secretion by inhibiting the electrical activity of the GnRH pulse generator [37], without the mediation of endogenous opioid peptide neurons [38]. Altogether, we assume that the icv injection of NMU in the present study results in the activation of CRH neurons which in turn inhibits the pulsatile GnRH secretion.
In the present study, the inhibitory action of NMU on the pulsatile LH secretion was weak in OVX rats under the normal feeding condition, but was strong under the 48-h fasted condition. It thus appears that the 48-h fasting somehow enhances the inhibitory action of NMU on the pulsatile LH secretion, although the fasting per se had no effect. The absence of any effect of 48-h fasting observed in the present study is consistent with a previous report [39]. Hence, there might be a certain synergism between NMU and fasting with respect to the control of the LH secretion. The mechanism for this synergism is not clear at present. However, one possibility is that CRH secretion is exaggerated under the 48-h fasted condition since 48-h food deprivation increases the catecholaminergic input to the PVN, which also results in CRH secretion [40].
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neurons including GnRH neurons, though no evidence is available to support this at present.
Acknowledgments We are grateful to Dr. K. Wakabayashi of Gunma University School of Medicine and the NIDDK for providing radioimmunoassay materials.
References
Fig. 4. Effects of icv injection of aCSF (n ¼ 6) or NMU (n ¼ 7) on the pulse frequency and the pulse amplitude in OVX rats under the 48-h fasted condition. They are expressed as the mean of the first hour (designated as 1 under the column), second hour (2), and third hour (3). Icv injections were made after the first hour. * and ** indicate p < 0:05 and p < 0:0001 vs the value of the first hour, respectively. a indicates p < 0:01 vs the value of the first and second hours in aCSFinjected OVX rats. See the text for further statistical details.
Contradictory reports have appeared, however, as to what neurons mediate the inhibitory action of fasting, insulin-induced hypoglycemia, and 2-deoxyglucose-induced cellular glycopenia on the pulsatile LH secretion. It was reported, on the one hand, that CRH neurons were involved in the inhibition of pulsatile LH secretion by insulin-induced hypoglycemia in OVX rhesus monkeys [38] and by fasting in estrogen-primed OVX rats [40,41]. Treatment with CRH antagonist is reported to delay the inhibition of the GnRH pulse generator activity in response to insulin-induced hypoglycemia in OVX rhesus monkeys [42]. On the other hand, it was reported that CRH neurons were not involved in the inhibition of pulsatile LH secretion by insulin-induced hypoglycemia in OVX ewes [43] and by 2-deoxyglucose in OVX rats without estrogen replacement [41]. Thus, we cannot deny the possibility that NMU acts on other
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Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions Hong Quan, Toshiya Funabashi,* Miyako Furuta, and Fukuko Kimura Department of Neuroendocrinology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan Received 1 October 2003
Abstract We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 ll) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Neuromedin U; Intracerebroventricular injection; Pulsatile LH secretion; Ovariectomized rats; Fasting; Feeding condition; Food deprivation; Luteinizing hormone; Female rats
Neuromedin U (NMU), a 23-amino-acid peptide, is a brain–gut peptide originally isolated from the spinal cord in pigs and later in many other species [1,2]. NMU acts through two receptors named NMU1R and NMU2R. The expression of rat NMU1R is abundant in peripheral tissues, but is sparse in the brain [3–6]. The expression of rat NMU2R is, on the other hand, mostly restricted to specific regions in the brain, such as the paraventricular nucleus (PVN), along the wall of the third ventricle in the hypothalamus, and the hippocampus [3,7,8]. Although NMU was initially shown to play a role in smooth muscle contraction [1], the expression of NMU2R in the brain suggests that NMU also plays a physiological role in the brain. Indeed, NMU induces adrenocorticotropic hormone (ACTH) secretion [9], suggesting involvement in the control of stress responses. Recently, it was also reported that central administration of NMU suppresses food intake in rats [10–12]. In addition, NMU mRNA is expressed * Corresponding author. Fax: +81-45-787-2578. E-mail address: [email protected] (T. Funabashi).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.052
abundantly in the ventromedial hypothalamic area which is an important site for inducing satiety, and its level is reduced in rats fasted for 48 h [3]. Thus, NMU appears to act in the hypothalamus as a satiety factor. Many neuropeptides which influence feeding behavior also affect reproductive functions. For example, ghrelin and orexin, which induce food intake and act as orexigenic factors [13,14], suppress pulsatile luteinizing hormone (LH) secretion [15–17]. In contrast, leptin acts as a satiety factor [18–20] and reverses the inhibition of pulsatile LH secretion by fasting [21]. Glucagon-like peptide-1 also acts as a satiety factor [22], stimulating LH secretion but decreasing its own level in the hypothalamus of male rats fasted for 48 h, which indicates a possible association of the satiety factor with the low LH levels in hungry animals [23]. These reports indicate that a satiety factor can prevent fasting-induced suppression of the pulsatile LH secretion. Since NMU acts as a satiety factor [3,10,11], we wondered whether NMU restores the pulsatile LH secretion which would have been inhibited by fasting. We therefore examined whether central administration of NMU influenced the
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pulsatile LH secretion in normally feeding rats or in 48h fasted rats. We report here that, different from leptin, NMU suppresses the pulsatile LH secretion under the 48-h fasted condition.
Materials and methods Animals. Female rats of Wistar strain (Charles River, Yokohama, Japan) were obtained at 7 weeks of age and maintained under controlled lighting conditions (lights on 05:00–19:00 h) with food and water available ad libitum. They were ovariectomized (OVX) and, for the intracerebroventricular (icv) injection, stereotaxically implanted with a guide cannula into the third ventricle according to the atlas of Albe-Fessard et al. [24] (stereotaxic coordinates: A ¼ 6:0, V ¼ 2:0, and L ¼ 0:0) under sodium pentobarbital (31.5 mg/kg body weight) anesthesia as reported previously [25]. All animal housing and surgical procedures were carried out according to the Guidelines laid down by the Institutional Animal Care and Use Committee of the Yokohama City University, Graduate School of Medicine. Two to three weeks after the brain surgery, each rat was handled daily for 10 min 1 week before the blood sampling. They were divided into two experimental groups; in experiment 1, the rats had access to an unlimited supply of food (normal feeding condition) and in experiment 2, the rats were deprived of food for 48 h (48-h fasted condition). An intraatrial cannula was implanted through the jugular vein on the day before the blood sampling. The blood sampling, approximately 120 ll each, was performed under freely moving conditions at 6-min intervals from 09:00 to 12:00 h. An equal volume of heparinized saline (2 IU/ml) was injected after each sampling. After the first hour of blood sampling, rat NMU (Peptide Institute, Osaka, Japan) dissolved in artificial cerebrospinal fluid (aCSF, 1 nmol/3 ll) was injected into the third ventricle through the guide cannula using a Hamilton microsyringe. This dose of NMU has been shown to induce satiety in rats [10]. The same volume of aCSF was injected as a control. Further blood samplings were performed for the second and third hours. Thus, the rats were arranged in a total of four groups: OVX rats under the normal feeding condition injected with aCSF (aCSF-injected OVX rats, n ¼ 7) or NMU (NMU-injected OVX rats, n ¼ 7) for experiment 1; OVX rats under the 48-h fasted condition injected with aCSF (aCSF-injected OVX rats, n ¼ 6) or NMU (NMU-injected OVX rats, n ¼ 7) for experiment 2. In a preliminary experiment, we found that estrogen replacement at a dose which significantly potentiated the inhibitory effect of orexin on the pulsatile LH secretion [16] did not affect the inhibitory effect of NMU under the 48-h fasted condition. Therefore, we used OVX rats without estrogen replacement in the present study. Serum concentrations of LH were measured by double antibody radioimmunoassay with materials supplied by the NIDDK and generously donated by Dr. K. Wakabayashi (Gunma University, Maebashi, Japan). The reference standard for LH was NIDDK rat LH-RP-4. However, the amount of LH is expressed in terms of NIH LH-S1. LH was determined in a single assay for experiment 1 and experiment 2. The minimally detectable amount of LH (95% confi-
dence limits of buffer controls) was 0.12 ng/ml (experiment 1) and 0.27 ng/ml (experiment 2), and the intraassay coefficient of variation (CV) for LH was 7.6% (experiment 1) and 8.7% (experiment 2). Statistics. The LH pulse detection was based on methods reported previously [26,27]. The analysis of LH pulsatility included the determination of pulse frequency (number of LH pulses in each of the first, second, and third hours) and pulse amplitude (difference between the peak and the pre-peak nadir) in each of the first, second, and third hours. The pulse amplitude was averaged for each of the 3 h in each rat and then mean amplitude was calculated for each group. In addition, the interpulse interval was calculated before (mean interpulse interval for the first hour) and after the injection (interpulse interval between the first and second pulse after the injection). If a first pulse after the injection did not appear within 24 min, which is the average interpulse interval without treatment (the first hour, Table 1), the latest pulse before the injection was defined as the first pulse after the injection (for example, Fig. 1 #15 and Fig. 3 #12). Analysis of variance (ANOVA) followed by Fisher’s PLSD post hoc comparison was used to test the statistical significance and significance was accepted at p < 0:05. Group data of serum LH concentrations over a 3-h period were also obtained by averaging values at the corresponding time. The data obtained this way were analyzed by paired t test. To this end, LH concentration was averaged for the first hour in each rat and compared to the corresponding time in each treatment group.
Results Experiment 1 Fig. 1 shows representative individual LH profiles and the mean LH concentration before and after icv injection of aCSF or NMU in OVX rats under the normal feeding condition. The pulsatile fluctuation of serum LH was evident during the blood sampling in aCSF-injected OVX rats, suggesting that icv injection of aCSF had no significant effect on the pulsatile LH secretion. NMU injection, on the other hand, seemed to inhibit the pulsatile LH secretion, since the serum LH concentration at the corresponding time after the injection was significantly lower than before the injection (Fig. 1, lower column). Two-way ANOVA, however, showed no significant effects of the treatment (p > 0:5, aCSF and NMU) and the time (p > 0:1, the first, second, and third hours) without the interaction (p > 0:1) on the pulse parameters, such as the frequency and the amplitude (Fig. 2). Two-way ANOVA of the interpulse interval, on the other hand, showed significance for the effects of both the treatment (p < 0:05, aCSF and NMU) and the time (p < 0:001, before and after the injection) with a significant interaction (p < 0:05).
Table 1 Effects of icv injection of aCSF or NMU on the interpulse interval in OVX rats under the normal feeding or the 48-h fasted condition Treatment
Normal feeding condition N
Before
After
N
Before
After
aCSF NMU
7 7
19.2 1.7 23.2 4.2
19.7 1.7 36.0 2.3*
6 7
25.0 1.8 20.7 1.8
28.0 1.3 54.9 6.7**
48-h fasted condition
* and ** indicate p < 0:005 and p < 0:001 vs the value before the injection, respectively. See the text for further statistical details.
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Fig. 1. Representative examples of effects of icv injection of aCSF (left panels) or NMU (right panels) on the serum concentration of LH in OVX rats under the normal feeding condition. The arrows indicate the time of icv injection of NMU or aCSF. Triangles indicate statistically defined LH pulses. The mean ( SEM) of LH concentrations at the corresponding time is shown in the lower panels. * indicates p < 0:05 vs. the value of the first hour. See the text for further statistical details.
Since the interaction was significant, we analyzed the data by one-way ANOVA (p < 0:001) and found by post hoc comparison that, in NMU-injected OVX rats, the interpulse interval after the injection was significantly larger than before the injection (Table 1, p < 0:005). Further, the interpulse interval in NMUinjected rats after the injection was significantly larger than in aCSF-injected rats before (p < 0:001) and after the injection (p < 0:001). ANOVA of the pulse, frequency (Fig. 2) and the interpulse interval (Table 1), indicated that, under the normal feeding condition, the pulsatile LH secretion was not markedly influenced by NMU injection (Fig. 2), but there was a statistically significant suppression by NMU injection in the interpulse interval (Table 1).
Experiment 2 In OVX rats under the 48-h fasted condition, icv injection of aCSF did not affect the pulsatile LH secretion, as shown in Fig. 3, since the pulsatile fluctuation of LH appeared to be comparable to the one in aCSF-injected OVX rats under the normal feeding condition. This suggests that the fasting condition used in the present study had no significant effect on the pulsatile LH secretion in OVX rats. ANOVA within the first hour of aCSF-injected OVX rats showed that there was no significant difference in the frequency (p > 0:5) or the amplitude (p > 0:5) between the normal feeding condition and the 48-h fasted condition. NMU injection, on the other hand, appeared to quickly suppress the pulsatile
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was no significant difference in the pulsatility between aCSF- and NMU-injected OVX rats before injection (p > 0:1). As shown in Table 1, two-way ANOVA of the interpulse interval showed a significant effect of the treatment (aCSF or NMU, P < 0:01) and the time (before and after the injection, p < 0:0001) with a significant interaction (p < 0:001). In one-way ANOVA (p < 0:0001) followed by post hoc comparison, the interpulse interval in NMU-injected OVX rats after the injection was significantly larger than in NMU-injected OVX rats before the injection (p < 0:001), aCSF-injected OVX rats before the injection (p < 0:001), and aCSF-injected OVX rats after the injection (p < 0:001). Finally, two-way ANOVA of the amplitude (Fig. 4) showed no significant effects of the treatment (p > 0:5, aCSF and NMU) and the time (p > 0:1, the first, second, and third hours) without the interaction (p > 0:1).
Discussion
Fig. 2. Effects of icv injection of aCSF (n ¼ 7) or NMU (n ¼ 7) on the pulse frequency and the pulse amplitude in OVX rats under the normal feeding condition. They are expressed as the mean of the first hour (designated as 1 under the column), second hour (2), and third hour (3). Icv injections were made after the first hour. No significant difference was found in these parameters.
LH secretion, as shown in Fig. 3. The serum LH concentration at the corresponding time after NMU injection was significantly lower than before the injection (Fig. 3, lower column). Two-way ANOVA of the frequency (Fig. 4) did not show any significant effect of the treatment (aCSF or NMU, P > 0:1), but showed a significant effect of the time (the first, second, and third hours, p < 0:01) with a significant interaction (p < 0:05). Since the interaction was significant, we analyzed the data by one-way ANOVA (p < 0:01). Post hoc comparison showed that the frequency in the second hour of NMU-injected OVX rats was significantly smaller than in the first hour of NMU-injected OVX rats (p < 0:0001) and aCSF-injected OVX rats (p < 0:005). In NMU-injected rats, the frequency in the third hour was significantly smaller than in the first hour (p < 0:05). Compared to aCSF-injected OVX rats at the corresponding time, the frequency was significantly smaller in the second hour in NMU-injected OVX rats (p < 0:01), but not in the first hour, suggesting that there
The present study shows for the first time that the central administration of NMU inhibits the pulsatile LH secretion and this effect is significantly enhanced under the 48-h fasted condition, which by itself does not markedly affect the pulsatile LH secretion. Since satiety factors such as leptin and glucagon-like peptide-1 stimulate the LH secretion [21,23], while orexinergic factors such as ghrelin, orexin, and melanin-concentrating hormone inhibit it [15,16,28], the inhibitory action of NMU on the LH secretion shown in the present study is in contrast to the action of satiety factors. It is therefore appears that NMU plays a different role in the control of the pulsatile LH secretion than other satiety-inducing peptides. In rats, the GnRH pulse generator in the hypothalamus governs the pulsatile LH secretion [29] and the frequency of LH pulses in general reflects the frequency of GnRH pulses [30]. Therefore, the observation that the frequency and the interpulse interval, but not the amplitude, of LH pulses was altered by NMU injection suggests that NMU acted on the hypothalamus but not on the anterior pituitary. If this is the case, the next question is how NMU inhibits the pulsatile GnRH secretion. It has been reported that a central administration of NMU stimulates ACTH and corticosterone secretions in male rats [31] and that NMU increases the release of CRH from hypothalamic explants in vitro [32]. Since NMU activates neurons in the PVN [32,33], it is likely that NMU induces the activation of CRH neurons in the PVN and, at least in part, neuroendocrine responses to NMU are mediated by CRH neurons. In fact, NMU-induced activation of locomotor activity, face-washing behavior, and grooming were partially abolished by pretreatment with an antagonist of CRH, a-helical CRH, or anti-CRH IgG, and these effects of
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Fig. 3. Representative examples of effects of icv injection of aCSF (left panels) or NMU (right panels) on the serum concentration of LH in OVX rats under the 48-h fasted condition. The arrows indicate the time of icv injection of NMU or aCSF. Triangles indicate statistically defined LH pulses. The mean ( SEM) of LH concentrations at the corresponding time is shown in the lower panels. * indicates p < 0:05 vs. the value of the first hour. See the text for further statistical details.
NMU were not observed in CRH knockout mice [34]. On the other hand, the involvement of CRH in the control of LH secretion has been suggested. It has been reported that the icv injection of CRH inhibited the pulsatile LH secretion in a dose-related manner in OVX female rats [35] irrespective of the presence of estrogen [36], indicating that the CRH system plays an inhibitory role in the LH secretion. It has also been reported that CRH inhibits the pulsatile LH secretion by inhibiting the electrical activity of the GnRH pulse generator [37], without the mediation of endogenous opioid peptide neurons [38]. Altogether, we assume that the icv injection of NMU in the present study results in the activation of CRH neurons which in turn inhibits the pulsatile GnRH secretion.
In the present study, the inhibitory action of NMU on the pulsatile LH secretion was weak in OVX rats under the normal feeding condition, but was strong under the 48-h fasted condition. It thus appears that the 48-h fasting somehow enhances the inhibitory action of NMU on the pulsatile LH secretion, although the fasting per se had no effect. The absence of any effect of 48-h fasting observed in the present study is consistent with a previous report [39]. Hence, there might be a certain synergism between NMU and fasting with respect to the control of the LH secretion. The mechanism for this synergism is not clear at present. However, one possibility is that CRH secretion is exaggerated under the 48-h fasted condition since 48-h food deprivation increases the catecholaminergic input to the PVN, which also results in CRH secretion [40].
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neurons including GnRH neurons, though no evidence is available to support this at present.
Acknowledgments We are grateful to Dr. K. Wakabayashi of Gunma University School of Medicine and the NIDDK for providing radioimmunoassay materials.
References
Fig. 4. Effects of icv injection of aCSF (n ¼ 6) or NMU (n ¼ 7) on the pulse frequency and the pulse amplitude in OVX rats under the 48-h fasted condition. They are expressed as the mean of the first hour (designated as 1 under the column), second hour (2), and third hour (3). Icv injections were made after the first hour. * and ** indicate p < 0:05 and p < 0:0001 vs the value of the first hour, respectively. a indicates p < 0:01 vs the value of the first and second hours in aCSFinjected OVX rats. See the text for further statistical details.
Contradictory reports have appeared, however, as to what neurons mediate the inhibitory action of fasting, insulin-induced hypoglycemia, and 2-deoxyglucose-induced cellular glycopenia on the pulsatile LH secretion. It was reported, on the one hand, that CRH neurons were involved in the inhibition of pulsatile LH secretion by insulin-induced hypoglycemia in OVX rhesus monkeys [38] and by fasting in estrogen-primed OVX rats [40,41]. Treatment with CRH antagonist is reported to delay the inhibition of the GnRH pulse generator activity in response to insulin-induced hypoglycemia in OVX rhesus monkeys [42]. On the other hand, it was reported that CRH neurons were not involved in the inhibition of pulsatile LH secretion by insulin-induced hypoglycemia in OVX ewes [43] and by 2-deoxyglucose in OVX rats without estrogen replacement [41]. Thus, we cannot deny the possibility that NMU acts on other
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