Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs.

The effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in sham-operated (Sham) and vestibular-lesioned (VL) mice were examined by in situ hybridization histochemistry. Corticotrophin-releasing hormone (CRH) in the paraventricular nucleus was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2 g environment compared with a 1 g environment. Significant decreases in pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript and significant increases in neuropeptide Y, agouti-related protein in the arcuate nucleus and orexin in the lateral hypothalamic area were observed in both Sham and VL mice. After 2 weeks of exposure, CRH and POMC were increased significantly in Sham but not in VL mice. After 8 weeks of exposure, the hypothalamic feeding-related neuropeptides were comparable between Sham and VL mice. These results suggest that the hypothalamic feeding-related neuropeptides may be affected during the exposed duration of hypergravity via vestibular inputs.

Involvement of central nesfatin-1 neurons on oxytocin-induced feeding suppression in rats.

Peripheral anorectic hormones, such as peptide YY (PYY) and oxytocin (OXT), suppress food intake. A newly identified anorectic neuropeptide, nesfatin-1, is synthesized in both peripheral tissue and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. Here, we examined the effects of intraperitoneal (ip) administration of PYY3-36, OXT, and OXT analog, on nesfatin-1-immunoreactive (ir) neurons in the rat hypothalamus and brainstem, using Fos double fluorescence-immunohistochemistry. The ip administration of OXT and OXT analog significantly increased the number of nesfatin-1-ir neurons expressing Fos-ir in the paraventricular nucleus, the arcuate nucleus, and the nucleus tractus solitarius, but not in the supraoptic nucleus, the lateral hypothalamic area, and the area postrema. No differences in the percentage of nesfatin-1-ir neurons expressing Fos in the nuclei of the hypothalamus and brainstem were observed, between rats treated with vehicle or those treated with PYY3-36. The decreased food intake, induced by OXT and OXT analog, was attenuated significantly by pretreatment with intracerebroventricular administration of antisense nesfatin-1. These results suggested that nesfatin-1-expressing neurons in the hypothalamus and brainstem may play a role in sensing the peripheral level of OXT and its suppression of feeding in rats.

Comparison of the induction of c-fos-eGFP and Fos protein in the rat spinal cord and hypothalamus resulting from subcutaneous capsaicin or formalin injection.

We evaluated whether a c-fos-enhanced green fluorescent protein (eGFP) transgenic rat line, which expresses the c-fos and eGFP fusion gene, can be useful for the study of nociceptive pathways and processing. Capsaicin solution (15%) or formalin (5%) was subcutaneously injected bilaterally into the hind paws (100µL per each paw) of adult male c-fos-eGFP transgenic or wild-type rats. Control rats were injected with ethanol or physiological saline respectively. Transgenic and wild-type rats were perfused at 1.5, 3 and 6h post injection, with some transgenic rats being perfused 24h post injection. A comparison of eGFP in transgenic rats and Fos-like immunoreactivity (LI) in wild-type rats was made in the dorsal spinal cord, paraventricular nucleus (PVN) and supraoptic nucleus (SON). Oxytocin-LI (OXT-LI) was carried out to examine the activation of OXT neurons in the PVN and SON. Following capsaicin or formalin treatment, eGFP was maximally expressed at 6h in the spinal cord and 3h in the PVN and SON, whereas, Fos-LI was maximally expressed at 1.5h in all the regions we analyzed. Induction of eGFP in the OXT neurons was observed after capsaicin or formalin treatment, while Fos-LI in the OXT neurons was observed only after formalin treatment. These results demonstrate that the peak induction of c-fos-eGFP following exposure to acute nociceptive stimuli was delayed by around 1.5-4.5h, but more sensitive than endogenous Fos, suggesting that the c-fos-eGFP rat line can be useful for the study of nociceptive pathways and processing.

Development of ghrelin resistance in a cancer cachexia rat model using human gastric cancer-derived 85As2 cells and the palliative effects of the Kampo medicine rikkunshito on the model.

Cancer cachexia (CC) is a multifactorial disease characterized by decreased food intake and loss of body weight due to reduced musculature with or without loss of fat mass. Patients with gastric cancer have a high incidence of cachexia. We previously established a novel CC rat model induced by human gastric cancer-derived 85As2 cells in order to examine the pathophysiology of CC and identify potential therapeutics. In patients with CC, anorexia is often observed, despite elevation of ghrelin, suggesting that ghrelin resistance may develop in these patients. In this study, we aimed to clarify the occurrence of ghrelin resistance in CC rats accompanied by anorexia and we investigated whether rikkunshito (RKT), a traditional Japanese Kampo medicine that potentiates ghrelin signaling, ameliorated CC-related anorexia through alleviation of ghrelin resistance. 85As2-tumor-bearing rats developed severe CC symptoms, including anorexia and loss of body weight/musculature, with the latter symptoms being greater in cachectic rats than in non-tumor-bearing or pair-fed rats. CC rats showed poor responses to intraperitoneal injection of ghrelin. In CC rats, plasma ghrelin levels were elevated and hypothalamic anorexigenic peptide mRNA levels were decreased, whereas hypothalamic growth hormone secretagogue receptor (GHS-R) mRNA was not affected. In vitro, RKT directly enhanced ghrelin-induced GHS-R activation. RKT administrated orally for 7 days partly alleviated the poor response to ghrelin and ameliorated anorexia without affecting the elevation of plasma ghrelin levels in CC rats. The expression of hypothalamic orexigenic neuropeptide Y mRNA but not hypothalamic GHS-R mRNA was increased by RKT. Thus, the 85As2 cell-induced CC rat model developed ghrelin resistance, possibly contributing to anorexia and body weight loss. The mechanism through which RKT ameliorated anorexia in the CC rat model may involve alleviation of ghrelin resistance by enhancement of ghrelin signaling. These findings suggest that RKT may be a promising agent for the treatment of CC.

Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50µg/kg) and secretin (100µg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

The ethanol metabolite acetaldehyde induces water and salt intake via two distinct pathways in the central nervous system of rats.

The sensation of thirst experienced after heavy alcohol drinking is widely regarded as a consequence of ethanol (EtOH)-induced diuresis, but EtOH in high doses actually induces anti-diuresis. The present study was designed to investigate the introduction mechanism of water and salt intake after heavy alcohol drinking, focusing on action of acetaldehyde, a metabolite of EtOH and a toxic substance, using rats. The aldehyde dehydrogenase (ALDH) inhibitor cyanamide was used to mimic the effect of prolonged acetaldehyde exposure because acetaldehyde is quickly degraded by ALDH. Systemic administration of a high-dose of EtOH at 2.5 g/kg induced water and salt intake with anti-diuresis. Cyanamide enhanced the fluid intake following EtOH and acetaldehyde administration. Systemic administration of acetaldehyde with cyanamide suppressed blood pressure and increased plasma renin activity. Blockade of central angiotensin receptor AT1R suppressed the acetaldehyde-induced fluid intake and c-Fos expression in the circumventricular organs (CVOs), which form part of dipsogenic mechanism in the brain. In addition, central administration of acetaldehyde together with cyanamide selectively induced water but not salt intake without changes in blood pressure. In electrophysiological recordings from slice preparations, acetaldehyde specifically excited angiotensin-sensitive neurons in the CVO. These results suggest that acetaldehyde evokes the thirst sensation following heavy alcohol drinking, by two distinct and previously unsuspected mechanisms, independent of diuresis. First acetaldehyde indirectly activates AT1R in the dipsogenic centers via the peripheral renin-angiotensin system following the depressor response and induces both water and salt intake. Secondly acetaldehyde directly activates neurons in the dipsogenic centers and induces only water intake.

Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus.

Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6-24h and in the PVN at 6 and 12h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA.

Neuropeptide W-Induced Hypophagia is Mediated Through Corticotropin-Releasing Hormone-Containing Neurons.

Neuropeptide W (NPW), which was originally isolated from the porcine hypothalamus, has been identified as the endogenous ligand for both the NPBWR1 (GPR7) and NPBWR2 (GPR8) receptors. These receptors, which belong to the orphan G protein-coupled receptor (GPCR) family, share a high sequence homology with the opioid and somatostatin receptor families. NPW and NPBWR1 are widely distributed in the rat central nervous system (CNS). While the intracerebroventricular (i.c.v.) injection of NPW elevates plasma corticosterone levels, the intravenous administration of NPW in conjunction with a corticotropin-releasing hormone (CRH) antagonist blocks NPW-induced corticosterone secretion. It has been reported that NPW is involved in regulating the hypothalamus-pituitary-adrenal cortex (HPA) axis and that i.c.v. administration of NPW decreases feeding behavior. The aim of the present study was to ascertain if NPW's role in feeding regulation is mediated (or not) through corticotropin-releasing hormone (CRH)-containing neurons. We found that NPW-containing axon terminals make synapses with CRH-immunoreactive cell bodies and dendritic processes in the hypothalamic paraventricular nucleus (PVN). The central infusion of NPW significantly induced c-Fos expression in CRH-immunoreactive neurons in the mouse PVN, but not in vasopressin- or oxytocin-immunoreactive neurons. To determine if NPW regulates feeding behavior through CRH neurons, the feeding behavior of mice was studied following the i.c.v. administration NPW in the presence or absence of pretreatment with a CRH antagonist. While NPW administration decreased feeding activity, the CRH antagonist inhibited this effect. These results strongly suggest that NPW regulates feeding behavior through CRH neurons in the mouse brain.

New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line.

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

Effects of food deprivation on the hypothalamic feeding-regulating peptides gene expressions in serotonin depleted rats.

We examined the effects of serotonin (5-HT) depletion induced by peripheral injection of 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) on the expression of feeding-regulating peptides expressions by using in situ hybridization histochemistry in adult male Wistar rats. PCPA pretreatment had no significant effect on basal levels of oxytocin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), neuropeptide-Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin in the hypothalamus. Food deprivation for 48 h caused a significant decrease in CRH, TRH, POMC, and CART, and a significant increase in NPY, AgRP and MCH. After PCPA treatment, POMC and CART did not decrease despite food deprivation. NPY was significantly increased by food deprivation with PCPA, but was attenuated compared to food deprivation without PCPA. These results suggest that the serotonergic system in the hypothalamus may be involved in the gene expression of POMC, CART, and NPY related to feeding behavior.

The gene expression of the hypothalamic feeding-regulating peptides in cisplatin-induced anorexic rats.

Cisplatin has been widely used; however, various disadvantageous side effects afflict patients. Rikkunshito (RKT), a traditional Japanese herbal medicine, has been widely prescribed in Japan to improve anorexia; but the mechanisms are unknown. Here we studied whether RKT could improve anorexia induced by cisplatin and changes in feeding-regulating peptides in the hypothalamus in rats. Adult male rats were divided into 4 groups: water+saline (WS), water+cisplatin (WC), RKT+saline (RS), and RKT+cisplatin (RC) groups. Water or RKT (1g/kg) was intragastrically administered for 4 days, from day -1 to day 2, and saline or cisplatin (6mg/kg) was intraperitoneally (i.p.) administered at day 0. After i.p. administration, cumulative food intake, water intake, urine volume and body weight were measured. The rats were then decapitated, followed by removal of the brain, and feeding-regulating peptides in the hypothalamus were measured by in situ hybridization histochemistry. In the three-day measurements, there were no significant changes in cumulative water intake and urine volume. The body weight and cumulative food intake in WC significantly decreased compared to WS, whereas these were not observed in RC. Pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC) in WC significantly increased, and neuropeptide Y (NPY) in the ARC decreased compared to WS, whereas those in RS and RC were comparable to WS. These results suggest that RKT may have therapeutic potential for anorexia induced by cisplatin.

Similar changes of hypothalamic feeding-regulating peptides mRNAs and plasma leptin levels in PTHrP-, LIF-secreting tumors-induced cachectic rats and adjuvant arthritic rats.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in malignancy. However, it is difficult to explain the mechanism of anorexia/cachexia with PTHrP secretion in detail. Previously, we demonstrated that the expressions of orexigenic peptides increased and anorexigenic peptides decreased under cachectic conditions in rats carrying tumors secreting PTHrP. In this study, we investigated whether such changes in the expression of hypothalamic feeding-regulating peptides can be solely attributed to PTHrP or are a general response under cachectic conditions. Cachectic syndromes were induced in rats by: (i) inoculation of human lung cancer LC-6 cells that secreted PTHrP, (ii) inoculation of human melanoma SEKI cells that secrete not PTHrP but LIF1, (iii) injection of heat-killed Mycobacterium leading to arthritis (AA) and (iv) oral administration of a high dose of 1α,25(OH)(2)D(3) that resulted in hypercalcemia. The LC-6-bearing rats and AA rats were treated with or without anti-PTHrP antibody and indomethacin, respectively, and the expression of the hypothalamic feeding-regulating peptide mRNAs were examined by in situ hybridization histochemistry. The orexigenic peptide mRNAs, such as neuropeptide Y and agouti-related protein, were significantly increased, and that of anorexigenic peptide mRNAs, such as proopiomelanocortin, cocaine- and amphetamine-regulated transcript and corticotropin-releasing hormone were significantly decreased when they developed cachectic syndromes and AA. A high dose of 1α,25(OH)(2)D(3) caused hypercalcemia and body weight loss but did not affect the expression of hypothalamic feeding-regulating peptide mRNAs. The expressions of the hypothalamic feeding-regulating peptides change commonly in different chronic cachectic models without relating to serum calcium levels.

Centrally administered adrenomedullin 5 activates oxytocin-secreting neurons in the hypothalamus and elevates plasma oxytocin level in rats.

We examined the effects of i.c.v. administration of adrenomedullin 5 (AM5) on the brain of conscious rats. We used porcine AM5 in the present study because rat AM5 has not been detected. We observed Fos-like immunoreactivity (LI) in the hypothalamus and brainstem of conscious rats after i.c.v. administration of AM5 (2 nmol/rat). Fos-LI, measured at 90 min post-AM5 injection, was observed in various brain areas, including the supraoptic (SON) and the paraventricular nuclei (PVN). Dual immunostaining for Fos/oxytocin (OXT) and Fos/arginine vasopressin (AVP) revealed that OXT-LI neurones predominantly colocalized Fos-LI compared with AVP-LI neurones in the SON and the PVN. Plasma OXT levels were significantly increased 5 min after i.c.v. administration of AM5 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 15 and 30 min without changes in plasma AVP levels at any time. In situ hybridization histochemistry showed that i.c.v. administration of AM5 (0.2, 1 and 2 nmol/rat) caused a marked induction of the expression of the c-fos gene in the SON and the PVN. This induction was significantly but not completely reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37; 3 nmol/rat) and the AM receptor antagonist AM-(22-52; 27 nmol/rat). Although porcine AM5 has not been detected yet in the brain, these results suggest that centrally administered porcine AM5 may activate OXT-secreting neurosecretory cells in the hypothalamus partly through AM/CGRP receptors and elicit secretion of OXT into the systemic circulation in conscious rats.

Adrenomedullin 2/intermedin-like immunoreactivity in the hypothalamus and brainstem of rats.

Adrenomedullin 2 (AM2) (identical to intermedin)-like immunoreactivity (LI) was examined in the rat brain by immunohistochemistry after intracerebroventricular administration of colchicine (100 microg/rat) and chronic salt loading (2% saline to drink) for 5 days. In both vehicle-treated and euhydrated rats, AM2-LI neurons were observed in the hypothalamus and brainstem, including in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the ventromedial hypothalamic nucleus, the arcuate nucleus, the locus coeruleus, the nucleus of the tractus solitarius and the nucleus ambiguus. In colchicine-treated and salt loaded rats, AM2-LI neurons were visualized more strongly in the SON and the magnocellular part of the PVN than in those in each control. Some AM2-LI neurons appeared in the parvocellular part of the PVN in the colchicine-treated but not salt loaded rats. AM2-LI in the other areas of the hypothalamus and brainstem did not change after colchicine-treatment and chronic salt loading. These results suggest that AM2/intermedin in the hypothalamus and brainstem may play roles on neuroendocrine and autonomic functions, such as water/salt balance, in rats.

Downregulation of prolactin-releasing peptide gene expression in the hypothalamus and brainstem of diabetic rats.

We investigated the prolactin-releasing peptide (PrRP) mRNA levels in the hypothalamus and brainstem of streptozotocin (STZ)-induced diabetic rats and fa/fa Zucker diabetic rats, using in situ hybridization histochemistry. PrRP mRNA levels in the hypothalamus and brainstem of STZ-induced diabetic rats were significantly reduced in comparison with those of control rats. PrRP mRNA levels in the diabetic rats were reversed by both insulin and leptin. PrRP mRNA levels in the fa/fa diabetic rats were significantly reduced in comparison with those of Fa/? rats. PrRP mRNA levels in the fa/fa diabetic rats were significantly increased by insulin-treatment, but did not reach control levels in the Fa/? rats. We also investigated the effect of restraint stress on PrRP mRNA levels in STZ-induced diabetic rats. The PrRP mRNA levels in the control and the STZ-induced diabetic rats increased significantly after restraint stress. The diabetic condition and insulin-treatment may affect the regulation of PrRP gene expression via leptin and other factors, such as plasma glucose level. The diabetic condition may not impair the role of PrRP as a stress mediator.

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats.

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM(22-52) and calcitonin gene-related peptide (CGRP) antagonist, CGRP(8-37) on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM(22-52) (27 nmol/rat) and CGRP(8-37) (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM(22-52)+CGRP(8-37) could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.

Parathyroid hormone-related protein induces cachectic syndromes without directly modulating the expression of hypothalamic feeding-regulating peptides.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. EXPERIMENTAL DESIGN: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating peptide genes and serum leptin levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. RESULTS AND CONCLUSION: The mRNA levels for the orexigenic peptides, such as the agouti-related protein and the neuropeptide Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the proopiomelanocortin in the Arc, the cocaine and amphetamine-regulated transcript in the Arc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for orexin. Thus, PTHrP induces HHM and concurrent cachectic syndromes by mechanisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.

The short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons but not arginine vasopressin-secreting neurons in the hypothalamus of rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-neurohypophyseal system were examined in rats. Plasma oxytocin (OXT) levels were significantly increased 15-60 min after i.p. administration of 2-B4O (100 mg/kg), whereas plasma arginine vasopressin (AVP) did not change. Dual immunostaining revealed that Fos-like immunoreactivity (LI) was predominantly observed in OXT-secreting neurons in the paraventricular (PVN) and the supraoptic nuclei (SON) 120 min after i.p. administration of 2-B4O. In addition, many Fos-LI neurons were observed in the nucleus of the tractus solitarius (NTS) after i.p. administration of 2-B4O. These results suggest that a peripherally administered high dose of 2-B4O activates OXT-secreting neurons in the hypothalamus through activation of NTS neurons, possibly as a result of a stress response.