Weaning stage hyperglycemia induces glucose-insensitivity in arcuate POMC neurons and hyperphagia in type 2 diabetic GK rats.

Hyperphagia triggers and accelerates diabetes, and prevents proper dietary control of glycemia. Inversely, the impact of hyperglycemia on hyperphagia and possible mechanistic cause common for these two metabolic disorders in type 2 diabetes are less defined. The present study examined the precise developmental process of hyperglycemia and hyperphagia and explored the alterations in the hypothalamic arcuate nucleus (ARC), the primary feeding and metabolic center, in Goto-Kakizaki (GK) rats with type 2 diabetes and nearly normal body weight. At mid 3 to 4 weeks of age, GK rats first exhibited hyperglycemia, and then hyperphagia and reduced mRNA expressions for anorexigenic pro-opiomelanocortin (POMC) and glucokinase in ARC. Furthermore, [Ca2+]i responses to high glucose in ARC POMC neurons were impaired in GK rats at 4 weeks. Treating GK rats from early 3 to mid 6 weeks of age with an anti-diabetic medicine miglitol not only suppressed hyperglycemia but ameliorated hyperphagia and restored POMC mRNA expression in ARC. These results suggest that the early hyperglycemia occurring in weaning period may lead to impaired glucose sensing and neuronal activity of POMC neurons, and thereby induce hyperphagia in GK rats. Correction of hyperglycemia in the early period may prevent and/or ameliorate the progression of hyperphagia in type 2 diabetes.

Unilateral lesion increases oestrogen receptor α expression in the intact side of the ventromedial hypothalamic nucleus in ovariectomised rats.

To determine the relationship between the right and left sides of the ventrolateral ventromedial hypothalamic nucleus (vlVMN) in regulating the expression of oestrogen receptor (ER)α, the unilateral vlVMN was lesioned and the number of ERα-immunoreactive cells and the ERα mRNA level in the intact side of the vlVMN and arcuate nucleus (ARC) were measured in ovariectomised rats. Twenty-four hours after lesioning, brain samples were collected for analysis of ERα expression by immunohistochemistry and the real-time reverse transcriptase-polymerase chain reaction. The number of ERα-immunoreactive cells in the intact side of the vlVMN but not the ARC in the unilateral lesioned group was significantly higher than that in the control or sham-lesioned group. Expression levels of ERα mRNA in the intact side of the vlVMN but not the ARC in unilateral lesioned rats were significantly higher than those in the sham-lesioned group. Of transcript variants with alternative 5'-untranslated regions (0S, 0N, 0, 0T and E1), the ERα 0 transcript level was significantly increased. These results indicate that unilateral damage of vlVMN induces an increase in ERα in the intact side by increasing ERα transcription in a promoter-specific manner. The findings also suggest the existence of new neuroendocrine control system between the right and left sides for the expression of ERα in the vlVMN.

Long-term infusion of brain-derived neurotrophic factor reduces food intake and body weight via a corticotrophin-releasing hormone pathway in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) has been implicated in learning, depression and energy metabolism. However, the neuronal mechanisms underlying the effects of BDNF on energy metabolism remain unclear. The present study aimed to elucidate the neuronal pathways by which BDNF controls feeding behaviour and energy balance. Using an osmotic mini-pump, BDNF or control artificial cerebrospinal fluid was infused i.c.v. at the lateral ventricle or into the paraventricular nucleus of the hypothalamus (PVN) for 12 days. Intracerebroventricular BDNF up-regulated mRNA expression of corticotrophin-releasing hormone (CRH) and urocortin in the PVN. TrkB, the receptor for BDNF, was expressed in the PVN neurones, including those containing CRH. Both i.c.v. and intra-PVN-administered BDNF decreased food intake and body weight. These effects of BDNF on food intake and body weight were counteracted by the co-administration of alpha-helical-CRH, an antagonist for the CRH and urocortin receptors CRH-R1/R2, and partly attenuated by a selective antagonist for CRH-R2 but not CRH-R1. Intracerebroventricular BDNF also decreased the subcutaneous and visceral fat mass, adipocyte size and serum triglyceride levels, which were all attenuated by alpha-helical-CRH. Furthermore, BDNF decreased the respiratory quotient and raised rectal temperature, which were counteracted by alpha-helical-CRH. These results indicate that the CRH-urocortin-CRH-R2 pathway in the PVN and connected areas mediates the long-term effects of BDNF to depress feeding and promote lipolysis.

Young adult-specific hyperphagia in diabetic Goto-kakizaki rats is associated with leptin resistance and elevation of neuropeptide Y mRNA in the arcuate nucleus.

The present study aimed to examine whether hyperphagia, which is frequently observed in type 1 diabetic patients and model animals, also occurs in type 2 diabetic Goto-Kakizaki (GK) rats and, if so, to explore underlying abnormalities in the hypothalamus. GK rats at postnatal weeks 6-12, compared to control Wistar rats, exhibited hyperphagia, hyperglycaemia, hyperleptinemia and increased visceral fat accumulation, whereas body weight was unaltered. The ability of leptin to suppress feeding was reduced in GK rats compared to Wistar rats of these ages. In GK rats, leptin-induced phosphorylation of signal transducer and activator of transcription 3 was significantly reduced in the cells of the hypothalamic arcuate nucleus (ARC), but not of the ventromedial hypothalamus, whereas the mRNA level of functional leptin receptor was unaltered. By real-time polymerase chain reaction and in situ hybridisation, mRNA levels of neuropeptide Y, but not pro-opiomelanocortin and galanin-like peptide, were significantly increased in the ARC of GK rats at 11 weeks, but not 26 weeks. Following i.c.v. injection of a NPY Y1 antagonist, 1229U91, the amount of food intake in GK rats was indistinguishable from that in Wistar rats, thus eliminating the hyperphagia of GK rats. These results demonstrate that young adult GK rats display hyperphagia in association with leptin resistance and increased NPY mRNA level in the ARC.

Overexpression of gastrin-releasing peptide receptor induced layer disorganization in brain.

Gastrin-releasing peptide-preferring and neuromedin B-preferring receptors, members of the bombesin-like peptide receptor subfamily, are reported to regulate proliferation, migration and differentiation. Since they are expressed in developing brain, we postulated that the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors might be involved in normal brain development. Here we examined the effects of the overexpressions of the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors on chick brain development in vivo using a retrovirus. In the overexpressed exogenous gastrin-releasing peptide-preferring receptor brain, we found laminar disorganization in the telencephalon, tectum and particularly in the cerebellum with severe atrophy. Processes of the radial glial cells in the telencephalon and optic tectum, as well as the projections of the Bergmann glia in the cerebellum were distorted, which might disturb normal cell migration. Despite the atrophy of the cerebellum, densely-stained proliferating cell nuclear antigen- and phospho-histone H3-positive cells increased in number. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells also increased in the cerebellum, suggesting that the ectopically proliferating cells were subjected to apoptosis. Glial fibrillary acidic protein-positive cells also increased in the hyperpallium accessorium and in the outer layers of the tectum. We also found smaller and spindle-shaped cells which resembled undifferentiated embryonic tumor cells. On the other hand, the layer structures of the neuromedin B-preferring receptors overexpressed brain were well organized and developed, and the size of brain was generally enlarged. These results indicated that although the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors are involved in normal brain development, both receptors contribute and exert their effects differently.

Study of the neutron beam line shield design for JSNS.

The JSNS, a spallation neutron source of J-PARC (JAERI-KEK Joint Project of the High Intensity Proton Accelerator) has 23 neutron beam lines. In the present study, a database was formulated for an optimum shielding design using the MCNP-X code. The calculations involved two steps. In the first step, the neutron distributions were created in the typical neutron beam line with a model that included the spallation neutron source target. The neutron currents evaluated flowed from the duct into the duct wall which was the boundary source for the bulk shield surrounding the beam line. In the second step, bulk-shield calculations were performed for the various shielding materials (iron, concrete, heavy concrete and so on) used and their composites up to thicknesses of 3 m. The results were compared with each other. Composite material shields of iron and such hydrogeneous materials as polyethylene or concrete were more effective. A typical design was prepared for a beam line within 25 m distance from a moderator, as a sample.

Distributions of two chicken bombesin receptors, bombesin receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R) mRNAS in the chicken telencephalon.

Bombesin (BN)-like peptide receptors are known to be essential to the regulation of not only homeostasis, including feeding behavior, but also of emotional systems in mammal. Recently, two novel BN receptors, chicken BN-like peptide receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R), have been identified. Here, we report the localizations of these receptors' mRNAs in the chick brain through development using in situ hybridization. First, chBRS-3.5 mRNA signals were found in the dorsal ventricular ridge at embryonic day (ED) 9. Strong signals were observed in the hyperpallium accessorium, nidopallium and nucleus basorostralis pallii, and moderate signals were found in the hippocampus, cortex piriformis, hyperpallium intercalatum, area temporo-parieto-occipitalis, nucleus striae terminalis lateralis, nucleus olfactorius anterior and organum septi lateralis at ED16. This wide expression in the pallium persisted during posthatch periods. Abundant expressions in the hyperpallium, nidopallium, considered to be similar to the mammalian cortex, as well as in the hippocampus, indicate participation of these molecules in the processing of sensory information, motor function, learning and memory. Telencephalic areas devoid of chBRS-3.5 signals were the entopallium, arcopallium anterius, globus pallidus, nucleus intrapeduncularis, tuberculum olfactorius, nucleus septalis lateralis, hypothalamic and thalamic areas. In contrast to chBRS-3.5, chGRP-R mRNA signals were found in the pallidum at ED5 and 9. At ED16, chGRP-R mRNA signals were localized in the medial striatum and hypothalamus. GRP-R expression in the hypothalamic region was phylogenically conserved. Thus, chBRS-3.5 mRNA signals were distributed in a broader region and were more intense than chGRP-R mRNA. Taken together, chGRP-R and chBRS-3.5 mRNA occurred in similar regions of mammals that express GRP-R. BN/GRP-immunoreactive neurons and varicosities were found mainly in the pallium, especially in the hyperpallium accessorium and nidopallium, and this distribution coincided with that of chBRS-3.5 mRNA. This result suggests that the endogenous ligands for chBRS-3.5 were likely BN-like peptides produced in the pallium.

Potential role of glial cell line-derived neurotrophic factor receptors in Müller glial cells during light-induced retinal degeneration.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.

Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat.

Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i. c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 microg/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly lower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 microg of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen. Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.

Peripheral or central administration of motilin suppresses LH release in female rats: a novel role for motilin.

Motilin is secreted in a clear episodic pattern during fasting or during the interdigestive phase, but feeding promptly stops this secretory pattern, and plasma concentrations of motilin decrease. We have previously determined that fasting markedly suppresses pulsatile luteinizing hormone (LH) secretion in female rats in the presence of oestrogen. In the present study, we wished to learn if motilin may mediate the fasting-induced suppression of LH secretion by determining the effects of motilin administration on LH release and on food intake. Intravenous (i.v.) injection of motilin (37 nmol/rat) suppressed LH release and significantly decreased mean LH concentrations both in ovariectomized (OVX) and oestradiol-implanted ovariectomized (OVX+E2) rats. Food intake was significantly increased by i.v. motilin injection in OVX rats, but not in OVX+E2 rats. It is likely that motilin inhibits LH release via inhibition of the gonadotrophin-releasing hormone (GnRH)-releasing mechanism at the hypothalamic level, because motilin (3.7 nmol/rat) also suppressed LH secretion when centrally administered, and because LH release in i.v. motilin-treated rats increased in response to exogenous GnRH. These results suggest that motilin may be a peripheral signal for the suppression of LH secretion through central sensors.

Localization of glucokinase-like immunoreactivity in the rat lower brain stem: for possible location of brain glucose-sensing mechanisms.

Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic beta-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopositive cells comprise a part of a vast glucose-sensing mechanism in the brain.

Morphological characterization of relationship between gap junctions and gonadotropin releasing hormone nerve terminals in the rat median eminence.

The present study aimed to reveal possible morphological relationships between gonadotropin-releasing hormone (GnRH) nerve terminals and gap junctions in the median eminence. Coronal brain sections from castrated male rats were dual immunostained with GnRH and either connexin 26, 32, or 43, and examined by confocal laser microscopy. Connexin 43-immunoreactive puncta were distributed between GnRH-immunoreactive fibers, and some of them were colocalized with GnRH-immunoreactivities. Dual immunostaining with connexin 43 and glial fibrillary acidic protein revealed that most of the puncta were located in astrocytes. At the immunoelectron microscopic level, connexin 43-immunoreactivities were mainly located on the plasma membranes of glial-like processes. Few connexin 26- or connexin 32-immunoreactivities were found in the median eminence. The present results indicate the possibility that gap junctions play a role in the GnRH release at the median eminence.

Prevention of inhibitory effect of dorsal raphe nucleus lesions on ovulation and LH surge by 5-HT 2A/2C receptor agonists in female rats.

In order to investigate the role of the dorsal raphe nucleus and the serotonergic system in the regulation of ovulation, the number of ova and plasma luteinizing hormone (LH) concentrations were measured in female rats after making lesions in this nucleus (DRL) and/or treatment with 5-hydroxytryptamine (5-HT) receptor agonists or antagonists. DRL or sham lesion was made on the afternoon of proestrous (12:00-14:00 h) under ether anesthesia and the number of ova in the oviduct was counted on the next estrous and diestrous morning. In some animals, blood samples were taken via the atrial cannula during the proestrous evening for the radioimmunoassay of LH. All intact control and sham-operated females ovulated and plasma LH increased between 19:00 and 21:00 h. In contrast, ovulation was seen in only 36% of DRL rats. LH surge did not occur in this group. However, 80% of DRL rats ovulated after treatment with (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride [(+/-)-DOI; a 5-HT 2A/2C receptor agonist] at 15:00 h on proestrous day. LH surge was also observed in the DRL rats with (+/-)-DOI. On the other hand, only 8% of DRL rats ovulated after treatment with buspirone (5-HT 1A receptor agonist). Furthermore, when mianserin (5-HT 2A/2C receptor antagonist) was administered at 16:00 h on proestrous day, ovulation was not seen in all rats without DRL. These results suggest that the dorsal raphe nucleus plays an important role in induction of LH surge and ovulation and the 5-HT 2A/2C receptor system is involved in this mechanism.

Inhibitory effect of neural transections of dorsal raphe nucleus on induction of nocturnal prolactin surge by vaginal stimulation in ovariectomized rats.

The effect of complete (CC), anterior (AC) or posterior (PC) cut of the dorsal raphe nucleus (DRn) on induction of the nocturnal prolactin (PRL) surge by electrical vaginal stimulation (VS) was investigated in ovariectomized rats. Plasma level of PRL was measured by radioimmunoassay before and after VS. The data revealed that PRL levels increased in early morning on the day following VS in the rats without brain surgery or with sham-operation. In contrast, the nocturnal PRL surge did not occur in the CC, AC, or PC rats. These results suggest that both the anterior and the posterior fibers of the DRn plays an important role in induction of nocturnal PRL surge by VS in ovariectomized rats.

Effects of chronic lesions in mesencephalic raphe nuclei on induction of pseudopregnancy.

The role of mesencephalic raphe nuclei in the induction of pseudopregnancy was investigated in female rats. The dorsal or median raphe nucleus lesions (DRL or MRL, respectively) were made by means of a radiofrequency lesion generator. Two or 3 weeks after the operation, in order to induce pseudopregnancy, the vagina was stimulated electrically on the day of proestrus or 1 mg/kg b.w. reserpine was injected on the day of diestrus I. Traumatization by passing thread to one uterine horn was performed to induce deciduoma 5 days after vaginal stimulation or 3 days after reserpine injection. As the results, decidual response was seen in most control and sham females in both vaginal stimulation and reserpine-treated groups. In contrast, incidences of deciduoma in DRL females with vaginal stimulation or reserpine-injection were significantly lower than those in control and sham groups. In the MRL females with either vaginal stimulation or reserpine-treatment, incidences of deciduoma were comparable to those of the control and sham operated groups. These results suggest that the dorsal raphe nucleus plays an important role in pseudopregnancy-inducing mechanisms in female rats.

Effect of deprivation of serotonin by p-chlorophenylalanine on induction and maintenance of pseudopregnancy in female rats.

The effect of serotonin synthesis inhibitor, p-chlorophenylalanine (PCPA), on induction and maintenance of pseudopregnancy as indicated by deciduoma formation was examined in female rats. Animals were injected with 1 mg/kg b.wt. of reserpine on the day of metestrus, and silk thread was passed through and placed in the left uterine horn 3 days after reserpine to induce deciduoma. PCPA (100 mg/kg b.wt.) was injected daily for 4 days before or after reserpine in 15 and 13 rats, respectively. A single injection of PCPA was administered before reserpine in nine females. In another group of rats (N = 16), instead of PCPA, saline was injected four times before reserpine. Nineteen female rats were treated with reserpine only as a control group. Results showed 89% of the control and 81.3% of the saline-treated females had massive deciduoma in traumatized uterine horn. In contrast, only 33.3% or 46.2% females with daily treatments of PCPA for 4 days before or after reserpine showed positive decidual reaction. In addition, 88.9% of females with single injection of PCPA possessed uterine horns with deciduoma. These results suggest that 4 days of treatment with PCPA eliminate induction and/or maintenance of pseudopregnancy. Thus, some levels of serotonin are required to induce and maintain pseudopregnancy.