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Endocrinology 143(9):3268 –3275 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2002-220268

Ghrelin Stimulates GH But Not Food Intake in Arcuate Nucleus Ablated Rats HIDEKI TAMURA, JUN KAMEGAI, TAKAKO SHIMIZU, SHINYA ISHII, HITOSHI SUGIHARA, SHINICHI OIKAWA

AND

Department of Medicine, Nippon Medical School, Tokyo 113-8603, Japan Ghrelin, an endogenous ligand for the GH secretagogue receptor 1a (GHS-R1a), was originally purified from the rat stomach. Ghrelin mRNA and peptide have also been detected in the hypothalamus and pituitary. Ghrelin is a novel acylated peptide that regulates GH release and energy metabolism. GHSR1a mRNA is expressed in the pituitary gland as well as in several areas of the brain including the hypothalamus. In this study, we examined whether ghrelin could stimulate GH secretion and feeding in chronic GHRH, neuropeptide Y, and agouti-related protein deficient rats that were neonatally treated with monosodium glutamate (MSG), which destroys the neurons in the hypothalamic arcuate nucleus (ARC). Intravenous (iv) administration of rat ghrelin (10 ␮g/kg body weight) increased plasma GH levels significantly in the normal adult male rats during a GH trough period of pulsatile GH secretion, while iv injection of ghrelin in MSG-treated rats resulted in a markedly attenuated GH response. When rat

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HE RELEASE OF the GH from the pituitary is regulated by at least two hypothalamic hormones: GHRH and somatostatin (SS) (1). In addition, an endogenous peptide ligand for the GH secretagogue receptor 1a (GHS-R1a), ghrelin, has been purified from the rat stomach and subsequently cloned (2). Ghrelin is a novel acylated peptide that contributes to the regulation of GH release and energy metabolism. Thus, ghrelin is thought to be the third physiological regulator of GH release. Ghrelin mRNA and peptide have also been detected in rat and human hypothalami and pituitaries (2– 4). Like the synthetic GHSs, ghrelin administered in combination with GHRH develops a synergistic potent GH release, and the GH-releasing activity of ghrelin in vivo is dependent on GHRH (5). However, in vitro GH release profiles from rat pituitary cells induced by ghrelin differ from the synthetic GHSs; costimulation with ghrelin and GHRH elicited neither a synergistic nor an additive GH response, whereas the synthetic GHS elicited an additive GH response with GHRH (6). Therefore, although the underlining mechanisms are not clear, the GH-releasing actions of ghrelin and synthetic GHS are not identical. GHS-R1a mRNA is expressed in the pituitary gland as well as in several areas of the brain including the hypothalamus (7). In the hypothalamus, GHS-R1a mRNA is expressed in neuropeptide Y (NPY)/agouti-related protein (AGRP), SS, GHRH and proopiomelanocortin neurons of the hypothaAbbreviations: AGRP, Agouti-related protein; ARC, arcuate nucleus; AUC, area under the curve; BW, body weight; GHS, GH secretagogue; GHS-R, GHS receptor; icv, intracerebroventricular; MSG, monosodium glutamate; NPY, neuropeptide Y; SS, somatostatin.

ghrelin (10 ␮g/rat) was administered intracerebroventricular (icv), plasma GH levels were increased comparably in normal control and MSG-treated rats. However, the GH release after icv injection of ghrelin was markedly diminished compared with that after iv administration of a small amount of ghrelin in normal control rats (icv: 10 ␮g/rat, iv: approximately 4.0 ␮g/rat), indicating that the GH-releasing activity of exogenous ghrelin is route dependent and at least in part via hypothalamic ARC. The icv administration of 1 ␮g of ghrelin increased significantly 4-h food intake in normal control, whereas the peptide did not increase food intake in MSG-treated rats, indicating that the feeding response to ghrelin requires intact ARC. Taken together, the primary action of ghrelin on appetite control and GH releasing activity is via the ARC even though it might act on another type of GHS-R besides GHS-R1a. (Endocrinology 143: 3268 –3275, 2002)

lamic arcuate nucleus (ARC) (8, 9). We and others have previously reported that central administration of ghrelin increase food intake mediated via activating NPY/AGRP neurons (10 –15). In addition, we and others have reported synthetic GHS induced c-fos expression, a marker of neuronal activity, in the hypothalamic GHRH, NPY, and other neurons of the hypothalamus (16, 17). These data indicated that the synthetic GHS and ghrelin could elicit their activity via GHS-R1a. However, GHS-R subtypes have been identified (18), suggesting that a still unknown receptor subtype may exist. Thus, there is a possibility that the action of ghrelin is through another type of GHS-R of the hypothalamic nucleus besides GHS-R1a. To further understand the mechanisms underlying the action of the exogenous administration of ghrelin, we have examined the site of action of ghrelin for its GH-releasing and feeding activity using chronic GHRH, NPY, and AGRP-deficient rats that were neonatally treated with monosodium glutamate (MSG) which destroys the neurons in the hypothalamic ARC. Materials and Methods Animals Neonatal Sprague Dawley rats received sc injections of MSG in doses of 4 mg/g body weight (BW), on d 1, 3, 5, 7, and 9 after birth. Control rats were treated with saline. At 4 wk of age, the pups were weaned, sexed, and housed in groups according to treatment. The rats were housed in air-conditioned animal quarters, with lights on from 0800 – 2000 h, and were provided with food and water ad libitum. All procedures were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and

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the protocol was approved by the Nippon Medical School Animal Care Research Committee.

Surgery At 12 wk of age, 2 wk before the study, some rats were anesthetized with ether and a 23-gauge stainless-steel cannula was implanted into right lateral ventricle using a stereotaxic apparatus, as previously described (19). The upper incisor bar was set 3.3 mm below the interauricular line and the bregma was taken as A-P zero. The cannula tip was placed at A ⫺0.9, L ⫺1.2, and V ⫺3.6 mm, and secured in place with dental acrylic. Only those rats whose cerebrospinal fluid overflowed through the cannula were used for the experiment. They were kept in individual cages and habituated by handling every day. Five days before the study, rats were provided with an indwelling right atrial cannula under ketamine and xylazine anesthesia for the undisturbed sampling of blood.

Experimental procedures In the first study, the effects of iv or intracerebroventricular (icv) administration of rat ghrelin (Peptide Institute, Inc., Osaka, Japan) on the plasma GH levels were determined in normal control and MSG-treated rats. Serial blood specimens (200 ␮l) were withdrawn via the indwelling right atrial cannula every 20 min manually from 800 –2000 h. Rat ghrelin was given iv (10 ␮g/kg BW) or icv (10 ␮g/10 ␮l of saline per rat) 6 h after the beginning of the blood sampling and blood specimens were obtained at the times indicated in the figures. The time of 1400 h was chosen because this time represents the typical trough periods of GH secretion in our laboratory. Red blood cells suspended in warm saline were returned every 60 min to maintain circulation. All blood specimens were immediately centrifuged and plasma was stored at –20 C until assayed for GH. The GH content was measured with a double-antibody RIA using materials supplied by NIDDK, NIH (Bethesda, MD), as previously described (20). All samples were assayed at the same time. All values are expressed as nanograms per milliliter in terms of the NIDDK reference preparation, rat GH-RP-2. In the second study, the effects of ghrelin administration on 4-h food intake were examined in normal control and MSG-treated rats. Our preliminary studies showed that iv administration of ghrelin (10 ␮g/kg) did not increase food intake in intact male rats for 4 h (2.4 ⫾ 0.5 g for ghrelin vs. 1.9 ⫾ 0.8 g for saline; n ⫽ 5; P ⬎ 0.05). Thus, in this study, we compared the effects of icv administration of ghrelin in MSG-treated rats and control rats. Rat ghrelin (1 ␮g/rat) or saline was injected centrally in freely feeding rats during the light phase (1000 –1100 h). Food intake was measured at 4 h after rat ghrelin was injected.

Histological examinations; in situ hybridization After the experiments, animals were anesthetized with pentobarbital (70 mg/kg, ip). Anesthetized rats were perfused with 2% paraformaldehyde, and the brain was kept overnight at 4 C in the same fixative containing 20% sucrose. Frozen coronal sections, 30 ␮m thick, were cut with a cryostat, mounted onto Silan-coated slides (Shin-Etsu Chemical Co., Tokyo, Japan), and air-dried. The medial basal hypothalamus from the rostral end of the third ventricle to the caudal end of the ARC was processed for in situ hybridization using 35S-labeled riboprobes for GHRH, NPY, and AGRP mRNA, as previously described (15). The hybridization signal in the brain slice was determined from the autoradiograms using an MCID image analysis system (Imaging Research, Inc., St. Catherines, Ontario, Canada), as previously described (15). The ARC on one side was enclosed by a circle with a 1.12-mm radius as a fixed window that covered almost the whole area of the nucleus. The relative optical density within the window was measured. The background was estimated by measuring the relative optical density within a window placed over another area of the hypothalamus. The anatomical equivalence of hypothalamic sections among animals was obtained by selecting slides with the aid of a rat brain atlas (21) and verifying the anatomical identifications by staining sections with cresyl violet. Six sections per animal were examined. Five rats were used for the analysis.

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Data analysis All data are presented as the mean ⫾ sem. The AUC depicting plasma GH concentration over 45 min (AUC45) was measured with a planimeter. Statistical analysis were carried out using the Student’s t test for two-group experiments and an ANOVA using Duncan’s New Multiple Range test for experiments containing three or more groups. P ⬍ 0.05 was considered significant.

Results Characterization of MSG-treated rats

As previously reported from our group (22), body weight and pituitary weight were impaired in MSG-treated rats at 14 wk of age (BW ⫽ 355.6 ⫾ 8.7 g for MSG vs. 415.0 ⫾ 8.4 g for saline; n ⫽ 5; P ⬍ 0.01, and pituitary weight ⫽ 6.24 ⫾ 0.07 mg for MSG vs. 8.93 ⫾ 0.39 mg for saline; n ⫽ 5; P ⬍ 0.01). The treatment with MSG reduced the hypothalamic gene expression for GHRH, NPY, and AGRP, to 17.5 ⫾ 5.6%, 6.6 ⫾ 1.6%, and 9.6 ⫾ 3.1% of normal control rats, respectively (P ⬍ 0.01; Figs. 1 and 2). Effects of iv or icv infusion of ghrelin on GH release in vivo

Figure 3 shows the representative plasma GH profiles with iv or icv administration of saline or ghrelin. The mean GH responses, plasma peak GH value, and AUC over 45 min after ghrelin administration are summarized in Figs. 4, 5, and 6, respectively. As shown in Fig. 3A, control rats given iv saline exhibited the typical pulsatile pattern of GH secretion. The iv administration of rat ghrelin (10 ␮g/kg BW) increased plasma GH levels significantly in the normal adult male rat during the GH trough period of pulsatile GH secretion (Figs. 3B and 4 – 6). On the other hand, as previously reported, treatment with MSG decreased the mean circulating GH concentrations, GH peak pulse amplitude and frequency (Fig. 3C). The iv injection of rat ghrelin in MSG-treated rats resulted in a significant increase of plasma GH levels (Figs. 3D and 4 – 6). However, iv injection of rat ghrelin in MSG-treated rats resulted in a markedly attenuated GH response compared with that of normal control rats (peak GH values ⫽ 51.3 ⫾ 9.9% of the control rats; n ⫽ 5; P ⬍ 0.01; Fig. 5, and AUC45 ⫽ 45.6 ⫾ 9.2% of the control rats; n ⫽ 5; P ⬍ 0.01; Fig. 6). In addition to this severe reduction in the absolute GH response, injection of ghrelin induced 52-fold increases of GH over baseline levels in MSG-treated rats, whereas the peptide induced 107-fold increases in control rats. When rat ghrelin (10 ␮g/rat) was administered centrally, plasma GH levels were increased comparably in normal control and MSG-treated rats (Fig. 3, F and H; and Figs. 4 – 6). However, the GH release after icv injection of ghrelin was markedly diminished compared with that after iv administration of a small amount of ghrelin in control and MSGtreated rats (Figs. 4 – 6). Effects of icv infusion of ghrelin on 4-h food intake

The icv administration of 1 ␮g of rat ghrelin significantly increased 4-h food intake in normal control rats (Fig. 7, 6.5 ⫾ 0.4 g for ghrelin vs. 1.4 ⫾ 0.9 g for saline; P ⬍ 0.01), whereas the peptide did not increase food intake in MSG-treated rats (Fig. 7, 5.3 ⫾ 0.5 g for ghrelin vs. 4.1 ⫾ 1.1 g for saline; P ⬎ 0.05).

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FIG. 1. Effect of neonatal monosodium glutamate (MSG) treatment on the hypothalamic mRNAs of adult male rats. Neonatal rats received sc injections of MSG in doses of 4 mg/g body weight, on d 1, 3, 5, 7, and 9 after birth. Control rats were treated with saline. Hypothalamic mRNA levels in the ARC were determined by in situ hybridization at 14 wk of age. The upper, middle, and lower panels show representative in situ hybridization histochemistry for GHRH, NPY, and AGRP mRNAs, as revealed by emulsion autoradiography in the ARC of normal control and MSGtreated rats. The scale bar indicates 500 ␮m.

Discussion

Injection of MSG into neonatal rodents cause specific lesions in the hypothalamic ARC, resulting in a 70 –90% destruction of neuronal cell bodies, including GHRH-, NPY-, and AGRP-containing neurons (23–26). Consistent with these previous reports, GHRH mRNA levels as well as NPY and AGRP mRNA levels were markedly diminished in this

experiment. Thus, MSG-treated rats in this study could be used as a model to study GHRH, NPY, and AGRP deficiency. The results of the present study show that neonatal treatment with MSG decreases the plasma GH response after iv administration of ghrelin. One possible explanation for this phenomenon is the decreased pituitary responsiveness of MSG-treated rats. GHRH is a primary stimulus for the syn-


FIG. 2. Effect of neonatal monosodium glutamate treatment on the hypothalamic mRNAs of adult male rats. The bar graph is a summary of the in situ hybridization histochemical data which is expressed as a percent of the vehicle-treated control group. Experimental details are indicated in Fig. 1. The data represent the mean ⫾ SEM (n ⫽ 5 animals/group). **, P ⬍ 0.01, compared with the vehicle-treated control group.

thesis and release of GH from pituitary somatotropes and, in fact, the pituitary weights of MSG-treated rats were significantly reduced compared with normal control rats in this experiment. However, investigations focusing on GHRHinduced GH release have demonstrated that pituitary responsiveness to GHRH dose not differ between MSG-treated rats and normal control rats when the data are expressed as a percent of basal GH values. Our group reported that a single injection of GHRH resulted in an 100-fold increase in circulating GH concentrations in both control and MSGtreated rats under pentobarbital anesthesia (22). It has also been reported that a bolus injection of GHRH resulted in a 20- to 30-fold increase in serum GH concentrations in conscious intact and MSG-lesioned rats (27). In this study, a single injection of ghrelin resulted in 52-fold increase in circulating GH concentrations in MSG-treated rats, whereas a bolus injection of ghrelin resulted in a 107-fold increase in plasma GH concentration in intact male rats. These data indicated that it is unlikely that pituitary unresponsiveness is the sole determinant of the diminished GH response after iv administration of ghrelin in MSG-treated rats. A second possible explanation for the diminished GH response after iv administration of ghrelin in MSG-treated rats is that the GH-releasing activity of ghrelin in vivo is dependent on an intact GHRH signaling system. These are consistent with the report that pretreatment with a GHRH antiserum or a GHRH antagonist substantially reduces the response to a subsequent ghrelin challenge as well as a synthetic GHS (5). Therefore, we could expect that the GH-releasing activity of the exogenous ghrelin requires, at least in part, the hypothalamic ARC, including the GHRH neurons. On the other hand, the present study shows that partial destruction of the major hypothalamic neuronal systems causes a truncation of the GH response after iv administration of ghrelin, although it is clearly still present. This suggests that these pathways are required for the full GH response to ghrelin, but even in their absence a partial response is possible, presumably due to a direct effect on the pituitary.


Although we have previously reported that central administration of ghrelin (1 ␮g/rat) did not alter the GH secretory pattern of adult male rats (12), the present study shows that when ghrelin (10 ␮g/rat) was administered centrally, plasma GH levels were increased in normal control rats during the GH trough period. These results are consistent with the previous report that a single icv administration of ghrelin to rats increased the plasma GH concentration dose dependently (28). In this previous report, the maximum GH response after a large dose of ghrelin was 100 –120 ng/ml, which is comparable to our present results. Because a large dose of ghrelin (10 ␮g) was needed to increase GH secretion after icv administration, one might speculate that ghrelin could have leaked into the hypothalamic-hypophyseal portal circulation after icv administration. However, we think that it is unlikely for the following reasons: 1) The volume of icv administration (10 ␮l) was the same for both treatments (1 ␮g and 10 ␮g); however, all 5 rats did not respond to 1 ␮g of ghrelin, whereas all 5 rats responded to 10 ␮g of ghrelin. 2) Histological examinations showed that there was at least no destruction in the medial basal hypothalamus. 3) When rat ghrelin (10 ␮g/rat) was administered centrally, plasma GH levels were increased comparably in normal control and MSG-treated rats. If any stimulation after icv administration could be the results of leakage into the portal circulation, MSG-treated rats should be less responsive than control rats due to the atrophic pituitaries. Despite these arguments, there might still be a possibility that the stimulation of GH secretion after icv administration is the results of leakage into the portal circulation and, thus, a direct effect on somatotropes. The GH release after icv injection of ghrelin was markedly diminished compared with that after iv administration of a small amount of ghrelin. In addition, central administration of pharmacological doses of ghrelin resulted in the plasma GH increases which were comparable in normal control rats and MSG-treated rats with an atrophic pituitary gland. Thus, the GH-releasing activity of exogenous ghrelin was route dependent and it effectively increased plasma GH levels through systemic injection. Furthermore, it should be pointed out that the ARC is positioned adjacent to the median eminence, where the blood-brain barrier is deficient, and, therefore, can be influenced by circulating hormones (29, 30). These observations could support the concept that the principal site of ghrelin synthesis is the stomach and not the hypothalamus, and the role of ghrelin is to regulate GH secretion via the endocrine activity of stomach ghrelin (5). However, further studies will be needed to conclude that circulating ghrelin arising from the stomach is the major regulatory pathway. In contrast to the GH-releasing activity, a small amount of icv ghrelin (1 ␮g/rat) increased food intake significantly in normal control rats. In MSG-treated rats, light-photoperiod 4-h food consumption tended to increase compared with normal control rats; however, 24-h food intake was significantly decreased compared with control rats (data not shown). These results are consistent with previous reports that MSG-treated rats eat food constantly, both in light and dark photoperiods, due to the damage to cells in the inner retina (31, 32). Recently, it has been reported that, when

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FIG. 3. Effect of iv or icv administration of ghrelin on plasma GH profiles in normal control and MSG-treated rats. Individual representative 6-h plasma GH profiles in an iv saline-infused control rat (A), an iv ghrelin-infused control rat (B), an iv saline-infused MSG-treated rat (C), an iv ghrelin-infused MSG-treated rat (D), an icv saline-infused control rat (E), an icv ghrelin-infused control rat (F), an icv saline-infused MSG-treated rat (G), and an icv ghrelin-infused MSG-treated rat (H). Rat ghrelin was given iv (10 ␮g/kg BW; approximately 4.0 ␮g/rat) or icv (10 ␮g/rat) at 1400, which was chosen because this time reflects the typical trough periods of GH secretion, and blood specimens were obtained at the times indicated in the figure. Note that each graph has a different scale.




FIG. 4. Mean plasma GH response to 10 ␮g/kg BW iv (A) and 10 ␮g/rat icv (B) administration of ghrelin at 1400 h in control and MSGtreated rats. The experimental details are indicated in Fig. 3. Note that ghrelin effectively increased plasma GH levels through systemic injection and that plasma GH levels were increased comparably in normal control and MSG-treated rats after icv injection of ghrelin. The values are the mean ⫾ SEM (n ⫽ 5 animals/ group). *, P ⬍ 0.05; **, P ⬍ 0.01, compared with the ghrelin-infused MSG-treated group.

FIG. 5. The plasma peak GH value after 10 ␮g/kg BW iv or 10 ␮g/rat icv administration of ghrelin at 1400 h in control and MSG-treated rats. The experimental details are indicated in Fig. 3. The values are the mean ⫾ SEM (n ⫽ 5 animals/group). *, P ⬍ 0.05, **, P ⬍ 0.01, compared with the saline-infused control group.

injected centrally, ghrelin increases food intake by activating NPY/AGRP-containing neurons (10 –15). In addition, it has been reported that the effect of ghrelin on food intake was blocked by pretreatment with a NPY Y1 receptor antagonist (14, 33). However, it has also been reported that the orexigenic effects of ghrelin are present in NPY-knockout mice (10). Our findings that ghrelin could not increase food intake in NPY- and AGRP-deficient rats are consistent with the idea that AGRP could compensate for the lack of NPY in NPYknockout animals (10, 34). In this study, the effects of exogenous administration of ghrelin on feeding and GH releasing activity were examined. Ghrelin was originally isolated from rat and human stomach. Ghrelin mRNA and peptide have also been detected in rat and human hypothalami and pituitaries. However, the role

of endogenous ghrelin has yet to be fully determined. Ghrelin is synthesized primarily in endocrine cells in the stomach and is present in the circulation. It has been reported that the plasma concentration of ghrelin after ip injection of the minimum effective dose (1 nmol/rat) to increase food intake was not significantly greater than plasma concentration seen after a 24-h fast, indicating that the stimulation of feeding by ghrelin could occur at plasma concentrations within the normal fasting range (35). On the other hand, circulating human plasma ghrelin shows a diurnal pattern with preprandial increases, postprandial decreases, and a maximal peak at 0100 h that resembles the 24-h profile of human plasma GH concentration (36, 37). These data suggest a possible physiological role for circulating ghrelin in the control of food intake and GH release. In addition, we have

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FIG. 6. The AUC depicting plasma GH concentrations over 45 min after 10 ␮g/kg BW iv or 10 ␮g/rat icv administration of ghrelin at 1400 h in the control and MSGtreated rats. The experimental details are indicated in Fig. 3. The values are the mean ⫾ SEM (n ⫽ 5 animals/ group). *, P ⬍ 0.05, **, P ⬍ 0.01, compared with salineinfused control group.

hypothalamic ghrelin has not been determined. Further studies will be necessary to investigate the physiological role of hypothalamic ghrelin in the regulation of feeding behavior and GH-releasing activity. In summary, we have shown that the GH-releasing activity of exogenous ghrelin was route dependent. We also demonstrated that the primary action of ghrelin on appetite control and GH releasing activity is at least in part via the hypothalamic ARC even through it might act on another type of GHS-R besides GHS-R1a. Acknowledgments We thank Ms. Masayo Asizawa for technical assistance.

FIG. 7. Effect of icv administration of ghrelin on the light photoperiod food consumption of freely feeding adult male control and MSGtreated rats. Rat ghrelin (1 ␮g/rat) or saline was injected in freely feeding rats during the light phase (between 1000 and 1100 h), and food consumption was measured 4 h later. The data represent the mean ⫾ SEM (n ⫽ 5 animals/group). **, P ⬍ 0.01, compared with the saline-treated control group.

previously shown that the infusion of GHRH increases the expression in the pituitary of the genes for ghrelin and GHS-R in freely-moving adult male rats, indicating that GHRH infusion activates the pituitary ghrelin/GHS-R signaling system that may influence the release of GH (4, 38). Therefore, in combination with circulating ghrelin, pituitary ghrelin could modulate the regulation of GH secretion by GHRH. In contrast, there is one published report on the presence of ghrelin in the hypothalamic ARC of colchicinetreated rats determined with immunohistochemistry (2). However, so far to our best knowledge, the regulation of

Received March 5, 2002. Accepted May 8, 2002. Address all correspondence and requests for reprints to: Jun Kamegai, M.D., Department of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113-8603, Japan. E-mail: [email protected]. This work was supported by a Grant-in-Aid for scientific research from the Japanese Ministry of Education, Science and Culture (to J.K.).

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