Foetal proglucagon processing in relation to adult appetite control: lessons from a transplantable rat glucagonoma with severe anorexia.

We have previously reported severe anorexia abruptly induced in rats 2-3 weeks after they have been transplanted subcutaneously with the glucagonoma MSL-G-AN. Vagotomy did not affect the time of onset and severity of anorexia, and the anorectic state resembles hunger with strongly elevated neuropeptide Y (NPY) mRNA levels in the nucleus arcuatus. We now show that circulating levels of bioactive glucagon-like peptide-1 (GLP-1) (7-36amide) start to increase above control levels exactly at the time of onset of anorexia. At this time-point, bioactive glucagon as well as total glucagon precursors and GLP-1 metabolites are already vastly elevated compared to controls. We further show that intravenous administration of very high concentrations of GLP-1 to hungry schedule-fed rats causes anorexia in a dose-dependent manner, which is blocked by the GLP-1 receptor antagonist exendin (9-39). GLP-1 (7-36amide) has a well-characterized anorectic effect but also causes taste aversion when administered centrally. The anorectic effect is blocked in rats treated neonatally by monosodium glutamate (MSG). We show that MSG treatment does not prevent the MSL-G-AN-induced anorexia, thereby suggesting a different type of anorectic function. We show a very strong component of taste aversion as anorectic rats, when presented to novel or known alternative food items, will resume normal feeding for 1 day, and then redevelop anorexia. We hypothetize that the anorexia in MSL-G-AN tumour-bearing rats correlates with a foetal processing pattern of proglucagon to both glucagon and GLP-1 (7-36amide), and is due to taste aversion. The sudden onset is characterized by a dramatic increase in circulating levels of biologically active GLP-1 (7-36amide), suggesting eventual saturation of proteolytic inactivation of its N-terminus.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake.

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Powerful inhibition of kainic acid seizures by neuropeptide Y via Y5-like receptors.

Neuropeptide Y (NPY) is widely distributed in interneurons of the central nervous system (CNS), including the hippocampus and cerebral cortex, in concentrations exceeding those of any other known neuropeptides. Sequence data comparing different species show that NPY is highly conserved. This suggests a critical role in regulation of regional neuronal excitability. Kainic acid, a glutamate agonist at kainic acid receptors, causes severe limbic motor seizures culminating in status epilepticus. We here report that NPY administered into the lateral ventricle is a powerful inhibitor of motor as well as electroencephalographic (EEG) seizures induced by kainic acid. This effect was mediated via receptors with a pharmacological profile similar to the recently cloned rat Y5 receptor. The present study is the first to demonstrate that NPY possesses anticonvulsant activity. This is consistent with the concept that NPY is an endogenous anticonvulsant and suggests that agonists acting at Y5-like receptors may constitute a novel group of drugs in antiepileptic therapy.

Identification of cannabinoid type 1 receptor expressing cocaine amphetamine-regulated transcript neurons in the rat hypothalamus and brainstem using in situ hybridization and immunohistochemistry.

Recent data have indicated that the neuropeptide cocaine amphetamine-regulated transcript (CART) may be a downstream mediator of the effect of CB1 receptor antagonist on appetite regulation. In order to identify possible interactions between CART and central CB1R expressing neurons, a detailed mapping of CART and CB1R expression and immunoreactivity in the brain was initiated. Single radioactive in situ hybridizations revealed a predominant overlap between CART and CB1R mRNA in hypothalamic and lower brainstem nuclei. Using double in situ hybridization, co-localization between CART and CB1R mRNA expressing neurons was observed to be most pronounced in the retrochiasmatic and lateral hypothalamic areas, as well as in all parts of the dorsal vagal complex. Further attempts to immunohistochemically characterize the distribution of CB1R were, however, deemed impossible as any of eight commercially available antibodies/antisera gave rise to non-specific staining patterns. Furthermore, the staining pattern obtained was not discriminate between CB1R knockout mice and wild type mice. Collectively, we demonstrate at the messenger level that CB1R expressing perikarya colocalize with CART expressing neurons in hypothalamic and brainstem areas known to be important in appetite control, whereas interactions at the protein level necessitate a demand for cautious interpretations of immunohistochemical results.

Combination of the insulin sensitizer, pioglitazone, and the long-acting GLP-1 human analog, liraglutide, exerts potent synergistic glucose-lowering efficacy in severely diabetic ZDF rats.

OBJECTIVE: Severe insulin resistance and impaired pancreatic beta-cell function are pathophysiological contributors to type 2 diabetes, and ideally, antihyperglycaemic strategies should address both. RESEARCH DESIGN AND METHODS: Therapeutic benefits of combining the long-acting human glucagon-like peptide-1 (GLP-1) analog, liraglutide (0.4 mg/kg/day), with insulin sensitizer, pioglitazone (10 mg/kg/day), were assessed in severely diabetic Zucker diabetic fatty rats for 42 days. Impact on glycaemic control was assessed by glycated haemoglobin (HbA(1C)) at day 28 and by oral glucose tolerance test at day 42. RESULTS: Liraglutide and pioglitazone synergistically improved glycaemic control as reflected by a marked decrease in HbA(1C) (liraglutide + pioglitazone: 4.8 +/- 0.3%; liraglutide: 8.8 +/- 0.6%; pioglitazone: 7.9 +/- 0.4%; vehicle: 9.7 +/- 0.3%) and improved oral glucose tolerance at day 42 (area under the curve; liraglutide + pioglitazone: 4244 +/- 445 mmol/l x min; liraglutide: 7164 +/- 187 mmol/l x min; pioglitazone: 7430 +/- 446 mmol/l x min; vehicle: 8093 +/- 139 mmol/l x min). A 24-h plasma glucose profile at day 38 was significantly decreased only in the liraglutide + pioglitazone group. In addition, 24-h insulin profile was significantly elevated only in the liraglutide + pioglitazone group. Liraglutide significantly decreased food intake alone and in combination with pioglitazone, while pioglitazone alone increased cumulated food intake. As a result, rats on liraglutide alone gained significantly less weight than vehicle-treated rats, whereas rats on pioglitazone alone gained significantly more body weight than vehicle-treated rats. However, combination therapy with liraglutide and pioglitazone caused the largest weight gain, probably reflecting marked improvement of energy balance because of reduction of glucosuria. CONCLUSIONS: Combination therapy with insulinotropic GLP-1 agonist liraglutide and insulin sensitizer, pioglitazone, improves glycaemic control above and beyond what would be expected from additive effects of the two antidiabetic agents.

Transplantable rat glucagonomas cause acute onset of severe anorexia and adipsia despite highly elevated NPY mRNA levels in the hypothalamic arcuate nucleus.

We have isolated a stable, transplantable, and small glucagonoma (MSL-G-AN) associated with abrupt onset of severe anorexia occurring 2-3 wk after subcutaneous transplantation. Before onset of anorexia, food consumption is comparable to untreated controls. Anorexia is followed by adipsia and weight loss, and progresses rapidly in severity, eventually resulting in reduction of food and water intake of 100 and 80%, respectively. During the anorectic phase, the rats eventually become hypoglycemic and hypothermic. The tumor-associated anorexia shows no sex difference, and is not affected by bilateral abdominal vagotomy, indicating a direct central effect. The adipose satiety factor leptin, known to suppress food intake by reducing hypothalamic neuropeptide Y (NPY) levels, was not found to be expressed by the tumor, and circulating leptin levels were reduced twofold in the anorectic phase. A highly significant increase in hypothalamic (arcuate nucleus) NPY mRNA levels was found in anorectic rats compared with control animals. Since elevated hypothalamic NPY is among the most potent stimulators of feeding and a characteristic of most animal models of hyperphagia, we conclude that the MSL-G-AN glucagonoma releases circulating factor(s) that overrides the hypothalamic NPY-ergic system, thereby eliminating the orexigenic effect of NPY. We hypothesize a possible central role of proglucagon-derived peptides in the observed anorexia.

Neurochemical characterization of hypothalamic cocaine- amphetamine-regulated transcript neurons.

The novel neuropeptide cocaine-amphetamine-regulated transcript (CART) is expressed in several hypothalamic regions and has recently been shown to be involved in the central control of food intake. To characterize the hypothalamic CART neurons and understand the physiological functions they might serve, we undertook an in situ hybridization and immunohistochemical study to examine distribution and neurochemical phenotype of these neurons. In situ hybridization studies showed abundant CART mRNA in the periventricular nucleus (PeV), the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), the arcuate nucleus (Arc), the zona incerta, and the lateral hypothalamic area. The distribution of CART-immunoreactive neurons as revealed by a monoclonal antibody raised against CART(41-89) displayed complete overlap with CART mRNA. Double immunohistochemistry showed co-existence of CART immunoreactivity (CART-IR) and somatostatin in some neurons of the PeV. In the magnocellular division of the PVN as well as the SON, CART-IR was demonstrated in both oxytocinergic and vasopressinergic perikarya. In the medial parvicellular region of the PVN a few CART-IR neurons co-localized galanin, but none was found to co-localize corticotropin-releasing hormone. In the Arc, almost all pro-opiomelanocortinergic neurons were shown to contain CART, whereas no co-localization of CART with NPY was found. In the lateral hypothalamic area nearly all CART neurons were found to contain melanin-concentrating hormone. The present data support a role for CART in neuroendocrine regulation. Most interestingly, CART is co-stored with neurotransmitters having both positive (melanin-concentrating hormone) as well as a negative (pro-opiomelanocortin) effect on food intake and energy balance.

Glucagon-like peptide 1(7-36) amide's central inhibition of feeding and peripheral inhibition of drinking are abolished by neonatal monosodium glutamate treatment.

In the rat, the glucagon-like peptide 1 (GLP-1)(7-36) amide inhibits neurones in the central nervous system responsible for food and water intake. GLP-1-induced inhibition of food intake may involve the hypothalamic arcuate nucleus, whereas rostral sensory circumventricular organs may be responsible for the inhibitory action of GLP-1 on drinking. To further investigate the role of these blood-brain-barrier-free areas in GLP-1-induced inhibition of ingestive behavior, neonatal Wistar rats were subjected to monosodium glutamate (MSG) treatment, which causes extensive damage to the arcuate nucleus as well as to parts of the sensory circumventricular organs. The inhibitory effect of GLP-1 on feeding induced by food deprivation was completely abolished in MSG-lesioned rats. This effect was not due to either a loss of sensitivity to anorectic agents or a loss of taste aversion because MSG-treated animals displayed normal anorectic responses to central administration of corticotropin-releasing factor and normal aversive responses to peripheral administration of both lithium chloride and D-amphetamine. In non-lesioned rats, neuropeptide Y (NPY)-induced feeding was significantly reduced by concomitant GLP-1 administration. In contrast, GLP-1 had no effect on NPY-induced feeding in MSG-lesioned rats, suggesting that the GLP-1 receptors that mediate inhibition of feeding are localized upstream to the NPY-sensitive neurones inducing feeding behavior. The inhibitory effect of GLP-1 on water intake was tested using an ANG II-elicited drinking paradigm. Central administration of GLP-1 inhibited ANG II drinking in both MSG-treated rats and their nontreated littermates. In contrast, peripheral administration of GLP-1 did not inhibit ANG II-induced drinking behavior in MSG-treated rats. Thus it is evident that centrally acting GLP-1 modulates feeding and drinking behavior via neurones sensitive to MSG lesioning in the arcuate nucleus and circumventricular organs, respectively.

Systemic administration of the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats.

Postprandial release of the incretin glucagon-like peptide-1 (GLP-1) has been suggested to act as an endogenous satiety factor in humans. In rats, however, the evidence for this is equivocal probably because of very high endogenous activity of the GLP-1 degrading enzyme dipeptidyl peptidase-IV. In the present study, we show that intravenously administered GLP-1 (100 and 500 microg/kg) decreases food intake for 60 min in hungry rats. This effect is pharmacologically specific as it is inhibited by previous administration of 100 microg/kg exendin(9-39), and biologically inactive GLP-1(1-37) had no effect on food intake when administered alone (500 microg/kg). Acute intravenous administration of GLP-1 also caused dose-dependent inhibition of water intake, and this effect was equally well abolished by previous administration of exendin(9-39). A profound increase in diuresis was observed after intravenous administration of both 100 and 500 microg/kg GLP-1. Using a novel long-acting injectable GLP-1 derivative, NN2211, the acute and subchronic anorectic potentials of GLP-1 and derivatives were studied in both normal rats and rats made obese by neonatal monosodium glutamate treatment (MSG). We showed previously that MSG-treated animals are insensitive to the anorectic effects of centrally administered GLP-1(7-37). Both normal and MSG-lesioned rats were randomly assigned to groups to receive NN2211 or vehicle. A single bolus injection of NN2211 caused profound dose-dependent inhibition of overnight food and water intake and increased diuresis in both normal and MSG-treated rats. Subchronic multiple dosing of NN2211 (200 microg/kg) twice daily for 10 days to normal and MSG-treated rats caused profound inhibition of food intake. The marked decrease in food intake was accompanied by reduced body weight in both groups, which at its lowest stabilized at approximately 85% of initial body weight. Initial excursions in water intake and diuresis were transient as they were normalized within a few days of treatment. Lowered plasma levels of triglycerides and leptin were observed during NN2211 treatment in both normal and MSG-treated obese rats. In a subsequent study, a 7-day NN2211 treatment period of normal rats ended with measurement of energy expenditure (EE) and body composition determined by indirect calorimetry and dual energy X-ray absorptiometry, respectively. Compared with vehicle-treated rats, NN2211 and pair-fed rats decreased their total EE corresponding to the observed weight loss, such that EE per weight unit of lean body mass was unaffected. Despite its initial impact on body fluid balance, NN2211 had no debilitating effects on body water homeostasis as confirmed by analysis of body composition, plasma electrolytes, and hematocrit. This is in contrast to pair-fed animals, which displayed hemoconcentration and tendency toward increased percentage of fat mass. The present series of experiments show that GLP-1 is fully capable of inhibiting food intake in rats via a peripherally accessible site. The loss in body weight is accompanied by decreased levels of circulating leptin indicative of loss of body fat. The profound weight loss caused by NN2211 treatment was without detrimental effects on body water homeostasis. Thus, long-acting GLP-1 derivatives may prove efficient as weight-reducing therapeutic agents for overweight patients with type 2 diabetes.

CART, a new anorectic peptide.

Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.

Binding of a iodinated substance P analog to a NK-1 receptor on isolated cell membranes from rat anterior pituitary.

The characteristics of binding sites for substance-P (SP) on rat anterior pituitary membranes were studied, using 125I-labeled Bolton-Hunter SP as a ligand. The binding of [125I]Bolton-Hunter SP was saturable and reversible, and reached a plateau of maximal binding after approximately 12 min of incubation. Scatchard and Hill analyses of specific binding revealed a single class of noninteracting binding sites with a high affinity (Kd = 0.72 nM) and a moderate density (binding capacity = 32.16 fmol/mg protein). The biologically active tachykinins, which, in addition to SP, consist of physalaemin, eledoisin, kassinin, neurokinin-A, and neurokinin-B, could inhibit the binding in a concentration-dependent manner. Based on titration curves, the IC50 values of the tachykinins were measured, and the receptor was classified as a NK-1 receptor. These results demonstrate that a population of specific SP-binding sites is present in rat anterior pituitary and further support evidence that this peptide has an influence on pituitary function.

Dehydration stimulates hypothalamic gene expression of histamine synthesis enzyme: importance for neuroendocrine regulation of vasopressin and oxytocin secretion.

Dehydration associated with hyperosmolality and decreased extracellular volume stimulates arginine vasopressin (AVP) and oxytocin (OT) secretion from magnocellular neurons of the hypothalamus. The effects of hyperosmolality and decreased extracellular volume on the magnocellular neurons are mainly indirect and seem to be mediated centrally via several neurotransmitters and neuropeptides. Because histamine (HA), which serves as a central neurotransmitter, releases AVP and OT from the neurohypophysis when administered centrally, we investigated the possible role of HA in dehydration-induced AVP and OT secretion. To do this, we studied 1) the effect of dehydration on messenger RNA (mRNA) expression of the HA synthesis enzyme, histidine decarboxylase (HDC), in the tuberomammillary nucleus of the hypothalamus; and 2) the effect of HA synthesis inhibition during dehydration on AVP and OT mRNA expression in the supraoptic nucleus of the hypothalamus as well as on plasma AVP and OT levels. Forty-eight hours of dehydration increased the mRNA level of HDC in the tuberomammillary nuclei, whereas 24 h of dehydration had no effect. Pretreatment with the HA synthesis inhibitor alpha-fluoromethylhistidine (alpha FMH) increased the expression of HDC mRNA in 24-h dehydrated rats, but had no effect in euhydrated rats. In rats dehydrated for 48 h, the already increased level of HDC mRNA was not increased further by alpha FMH. Twenty-four and 48 h of dehydration increased AVP and OT mRNA levels in the supraoptic nucleus. This effect was inhibited by alpha FMH pretreatment. Dehydration increased the plasma levels of AVP and OT to an extent which depended on the duration of dehydration. Pretreatment with alpha FMH inhibited the hormone responses to 24 h of dehydration, but did not affect the responses to 48 h of dehydration. Twenty-four and 48 h of dehydration had no significant effect on the contents of AVP and OT in the neurohypophysis, whereas pretreatment with alpha FMH combined with 48 h of dehydration led to depletion of AVP stores in the neurohypophysis. Based on the present findings, we conclude that hypothalamic histaminergic neurons are involved in the regulation of dehydration-induced stimulation of magnocellular AVP and OT neurons.

Histaminergic activation of the hypothalamic-pituitary-adrenal axis.

Centrally administered histamine (HA) stimulates the secretion of adenohypophysial POMC-derived peptides, which subsequently cause release of corticosterone. The effect of HA on POMC-derived peptide release is indirect, and it is possible that hypothalamic neurons containing corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), or oxytocin (OT) are involved in the mediation of this response. We studied the effect of HA on: 1) expression of CRH, AVP, and OT messenger RNA (mRNA) at the hypothalamic level; 2) expression of c-fos and POMC mRNA at the pituitary level; and 3) peripheral plasma levels of AVP, OT, ACTH, beta-endorphin (beta-END), and corticosterone. HA (270 nmol) infused intracerebroventricularly increased the expression of CRH, AVP, and OT mRNA in the paraventricular nucleus as well as that of OT mRNA in the supraoptic nucleus of the hypothalamus. At the pituitary level the expression of mRNA for c-fos and POMC increased in the anterior but not in the intermediate lobe in response to HA. Plasma levels of AVP, OT, ACTH, beta-END, and corticosterone all increased in response to central HA administration. Circulating levels of AVP and OT peaked after 5 min, ACTH and beta-END after 15 min, whereas corticosterone levels were highest after 30 min. In concert with our earlier discoveries, the present data support the hypothesis that HA-induced secretion of ACTH and beta-END is mediated via central activation of hypothalamic neuroendocrine neurons containing CRH, AVP, and/or OT.

Pituitary adenylate cyclase-activating peptide gene expression in corticotropin-releasing factor-containing parvicellular neurons of the rat hypothalamic paraventricular nucleus is induced by colchicine, but not by adrenalectomy, acute osmotic, ether, or restraint stress.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a newly discovered neuropeptide that is present in high amounts in hypothalamic neuroendocrine neurons and potently stimulates the accumulation of cAMP within cells of the anterior pituitary. We have employed several specific antisera recognizing different parts of the PACAP precursor to elucidate the distribution of PACAP-like immunoreactivities in the hypothalamic components of the hypothalamo-pituitary-adrenocortical axis in sections obtained from normal and colchicine-treated rats. Using immunohistochemistry with avidin-biotin-coupled peroxidase as a reporter system, high numbers of PACAP-immunoreactive perikarya were found in colchicine-pretreated rats in many of the parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVN). A few cells were also found in the magnocellular subdivisions of the nucleus, and a similar small population of cells was observed in the dorsolateral aspect of the supraoptic nucleus. Using indirect immunofluorescence, the relation between CRF- and PACAP-containing neurons in the various parvicellular subnuclei of the PVN was studied, and a high degree of colocalization was demonstrated in the neurons of the medial parvicellular part of PVN. To further study the functional implications of PACAP in the hypothalamo-pituitary-adrenocortical axis, we examined the expression of PACAP messenger RNA (mRNA) in the PVN in response to five different stimulatory paradigms that previously have been shown to stimulate CRF mRNA expression in the medial parvicellular part of the PVN. The stimulatory challenges of adrenalectomy, restraint stress, ip injection of hypertonic saline, ether stress, and intracerebroventricular injection of colchicine induced significant elevations of CRF mRNA expression in the medial parvicellular part of the PVN. In contrast, the expression of PACAP mRNA, which is hardly detectable within the medial parvicellular part of the PVN, was induced only by colchicine treatment (from undetectable levels to 177 +/- 21 dpm/g; mean +/- SEM), whereas PACAP mRNA remained undetectable in this region of the PVN after exposure to any of the other stimulatory paradigms. The onset of colchicine-induced PACAP mRNA expression in the PVN was rapid (3 h), and PACAP mRNA levels remained elevated throughout the 48-h observation period. Considering the different topography and connections of the parvicellular subnuclei of the PVN, the current observations suggest that PACAP present in parvicellular neurons of the PVN may act not only as a neuroendocrine transmitter/modulator in the hypothalamo-pituitary-adrenocortical axis, but also as transmitter mediating neurotransmission conveyed from the PVN to preganglionic neurons of the autonomic system.(ABSTRACT TRUNCATED AT 400 WORDS)

Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat.

We have recently shown that intracerebroventricular (i.c.v.) administration of the hypothalamic neuropeptide cocaine-amphetamine-regulated transcript (CART) inhibits food intake and induces the expression of c-fos in several nuclei involved in the regulation of food intake. A high number of CART-induced c-Fos-positive nuclei in the paraventricular nucleus of the hypothalamus prompted us to examine the effect of i.c.v. recombinant CART-(42-89) on activation of CRH-, oxytocin-, and vasopressin-synthesizing neuroendocrine cells in the paraventricular nucleus (PVN). In addition, plasma levels of glucose were examined after central administration of CART-(42-89). Seventy-six male Wistar rats were fitted with i.c.v. cannulas and singly housed under 12-h light, 12-h dark conditions. One week postsurgery the animals were injected i.c.v. in the morning with 0.5 microg recombinant CART-(42-89) or saline. Trunk blood was collected by decapitation at 0 (baseline), 10, 20, 40, 60, 120, or 240 min. CART caused a strong increase in circulating corticosterone that was significantly different from saline at 20, 40, 60, and 120 min postinjection (P<0.05). Furthermore, CART caused a transient rise in plasma oxytocin levels (P<0.05 at 10 and 20 min postinjection), whereas plasma vasopressin levels were unaffected by i.c.v. CART. Animals injected i.c.v. with CART showed a rise in blood glucose levels 10 min postinjection (P<0.05). To examine whether the stimulatory effect of i.c.v. CART on corticosterone and oxytocin secretion is caused by activation of paraventricular nucleus/supraoptic nucleus (PVN/SON) neuroendocrine neurons, we used c-Fos as a marker of neuronal activity. Animals injected with CART showed a strong increase in c-Fos-immunoreactive nuclei in the PVN. Double immunohistochemistry revealed that a high (89+/-0.4%) number of CRH-immunoreactive neurons in the PVN contained c-Fos after CART i.c.v.. c-Fos expression was also observed in oxytocinergic cells (in both magnocellular and parvicellular PVN neurons as well as in the supraoptic nuclei) 120 min after CART administration, whereas none of the vasopressinergic neurons contained c-Fos. Triple immunofluorescence microscopy revealed that CART-immunoreactive fibers closely apposed c-Fos-positive CRH neurons, suggestive of a direct action of CART on PVN CRH neurons. In summary, i.c.v. CART activates central CRH neurons as well as both magnocellular (presumably neurohypophysial) and parvicellular (presumably descending) oxytocinergic neurons of the PVN. The effect of CART on CRH neurons most likely leads to corticosterone secretion from the adrenal gland, which may contribute to the inhibitory effects of CART on feeding behavior.

Central administration of glucagon-like peptide-1 activates hypothalamic neuroendocrine neurons in the rat.

Within the central nervous system, glucagon-like peptide-1-(7-36) amide (GLP-1) acts as a transmitter, inhibiting feeding and drinking behavior. Hypothalamic neuroendocrine neurons are centrally involved in the regulatory mechanisms controlling these behaviors, and high densities of GLP-1 binding sites are present in the rat hypothalamus. In the present study we have, over a period of 4 h, followed the effect of centrally injected GLP-1 on plasma levels of the neurohypophysial hormones vasopressin and oxytocin. Plasma levels of corticosterone and glucose were also followed across time after central administration of GLP-1. In conscious, freely moving, and unstressed rats, central injection of GLP-1 significantly elevated plasma levels of vasopressin 15 and 30 min after administration (basal, 0.8 +/- 0.2 pg/ml; 15 min, 7.5 +/- 2.0 pg/ml; 30 min, 5.6 +/- 1.1 pg/ml; mean +/- SEM) and elevated corticosterone 15 min after administration (52 +/- 13 vs. 447 +/- 108 ng/ml, basal vs. 15 min; mean +/- SEM). In contrast, plasma oxytocin levels were unaffected by intracerebroventricular (icv) injections of GLP-1 over a period of 4 h after the injection. The animals given a central injection of GLP-1 developed transient hypoglycemia 20 min after the injection, which was fully restored to normal levels at 30 min. Furthermore, we used c-fos immunocytochemistry as an index of stimulated neuronal activity. The distribution and quantity of GLP-1-induced c-fos immunoreactivity were evaluated in a number of hypothalamic neuroendocrine areas, including the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei and the parvicellular neurons of the medial parvicellular subregion of the PVN. The number of c-fos-expressing nuclei in those areas was assessed 30, 60, and 90 min after icv administration of GLP-1. Intracerebroventricular injection of GLP-1 induced c-fos expression in the medial parvicellular subregion of the PVN as well as in magnocellular neurons of the PVN and SON. A slight induction of c-fos expression was seen in the arcuate nucleus and the nucleus of the solitary tract, including the area postrema. In contrast, the subfornical organ, which is a rostrally situated circumventricular organ, was free of c-fos-positive cells after central administration of GLP-1. When the GLP-1 antagonist exendin-(9-39) was given before the GLP-1, c-fos expression in these neuroendocrine areas was almost completely abolished, suggesting that the effect of GLP-1 on c-fos expression is mediated via specific receptors. A dual labeling immunocytochemical technique was used to identify the phenotypes of some of the neurons containing c-fos-immunoreactive nuclei. Approximately 80% of the CRH-positive neurons in the hypophysiotropic medial parvicellular part of the PVN coexpressed c-fos 90 min after icv GLP-1 administration. In contrast, very few (approximately 10%) of the vasopressinergic magnocellular neurons of the PVN/SON contained c-fos-positive nuclei, whereas approximately 38% of the magnocellular oxytocinergic neurons expressed c-fos-positive nuclei in response to GLP-1 administration. This study demonstrates that central administration of the anorectic neuropeptide GLP-1 activates the central CRH-containing neurons of the hypothalamo-pituitary-adrenocortical axis as well as oxytocinergic neurons of the hypothalamo-neurohypophysial tract. Therefore, we conclude that GLP-1 activates the hypothalamo-pituitary-adrenocortical axis primarily through stimulation of CRH neurons, and this activation may also be responsible for the inhibition of feeding behavior.

Histamine stimulates c-fos expression in hypothalamic vasopressin-, oxytocin-, and corticotropin-releasing hormone-containing neurons.

The stimulatory action of centrally administered histamine (HA) on secretion of the anterior pituitary hormones ACTH, beta-endorphin, and PRL is indirect, and previous studies have suggested that hypothalamic neurons containing CRH, arginine vasopressin (AVP), and oxytocin (OT) are involved in this response. We studied the effect of HA on neuronal activation in the hypothalamus by investigating the expression of c-fos, which is a protooncogene activated early when neurons are stimulated. The expression of c-fos was evaluated by detection of c-fos immunoreactivity (c-fos-IR) using immunohistochemistry and by measurement of c-fos mRNA using in situ hybridization techniques. In addition, the identity of the HA-stimulated neurons was investigated by dual antigen immunohistochemistry visualizing AVP-, OT-, or CRH-IR in the neurons showing increased c-fos expression. HA (270 nmol) infused intracerebroventricularly increased c-fos-IR in the hypothalamus, especially in the periventricular hypothalamic areas and certain hypothalamic nuclei, including the paraventricular nucleus (PVN) and supraoptic nucleus (SON). c-fos-immunoreactive nuclei were observed throughout the SON, whereas in the PVN, c-fos-IR was particularly pronounced in the subnuclei known to contain AVP, OT, and CRH neurons. Double labeling experiments confirmed that c-fos was expressed in AVP-, OT-, and CRH-immunoreactive as well as other neurons. In addition, HA intracerebroventricularly induced a moderate expression of c-fos-IR in the arcuate nucleus. In situ hybridization showed increased levels of c-fos mRNA in both the PVN and SON after HA infusion. We conclude that HA-induced secretion of ACTH, beta-endorphin, and PRL may be mediated via activation of hypothalamic AVP, OT, and CRH neurons.

Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland.

Neuropeptide-Y (NPY) is colocalized with vasopressin and oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial system, and a very high density of NPY-binding sites is present within the neurohypophysis. To investigate the possibility that NPY exerts a modulatory role on the release of neurohypophysial hormones, we have studied the actions of NPY on potassium-evoked release of vasopressin and oxytocin from the rat neurointermediate lobe in vitro. NPY dose-dependently potentiated vasopressin release evoked by high extracellular potassium (56 mM), with a maximal enhancement of 223% (10(-7) M NPY). A similar effect was obtained with the Y2-selective agonist NPY-(13-36). In contrast, no effect on the potassium-evoked release of oxytocin was observed at this concentration. In the absence of Ca2+ in the incubation medium, NPY did not potentiate vasopressin secretion, indicating that the effect of NPY on potassium-evoked secretion of neurohypophysial vasopressin is critically dependent on extracellular calcium ions. The number of neurohypophysial NPY-binding sites is drastically down-regulated in animals subjected to chronic osmotic stimulation. In the present study, it was observed that the potentiating effect of NPY on vasopressin secretion was completely abolished in neurointermediate lobes recovered from animals that had been drinking 2% NaCl for 12 days, reflecting the concomitant down-regulation of neurohypophysial NPY-binding sites observed during this state. Finally, it was confirmed that stimulation by high K+ significantly evoked the release of endogenous NPY from neurointermediate lobes of the pituitary gland. The present results provide evidence that NPY selectively and potently enhances evoked vasopressin secretion. Considering the coexistence of the two neuropeptides in magnocellular hypothalamo-neurohypophysial neurons, this action is likely to be part of an autostimulatory feedforward loop. NPY may be an important component in the mechanisms associated with the control of body fluid homeostasis.

Water deprivation increases the expression of pituitary adenylate cyclase-activating polypeptide gene in the rat subfornical organ.

The effect of water deprivation on the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in the rat subfornical organ (SFO), using a combination of immunohistochemistry and in situ hybridization histochemistry. In the euhydrated condition, PACAP-immunoreactivity (PACAP-IR) and the expression of PACAP gene was observed in the SFO. Water deprivation for 24 h and 48 h caused a significant increase in PACAP gene transcripts in the SFO, compared with euhydrated animals. Additionally, water deprivation for 48 h caused an increase in PACAP-IR. This increase of PACAP-IR was demonstrated in both nerve fibers and cell bodies. High correlation was found between the localization of PACAP-IR cell bodies and PACAP messenger RNA synthesizing cell bodies in the peripheral part of the SFO. These results suggest that PACAP in the SFO may play a role in the humoral and neural changes associated with the regulation of body fluid balance after water deprivation.