Paraventricular hypothalamic alpha-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects

alpha-Melanocyte-stimulating hormone (alpha-MSH) appears to play a tonic inhibitory role in feeding and energy storage. MTII, a specific synthetic MC3-R/MC4-R agonist, has similar effects on feeding in rats. The current studies demonstrate that PVN administration of alpha-MSH or MTII decreases nocturnal and NPY-stimulated food intake without causing aversive effects. Co-administration with NPY of 600 pmol alpha-MSH or 1 pmol MTII into the PVN caused a significant decrease in NPY-induced feeding. PVN administration of MTII or alpha-MSH at doses effective to suppress feeding did not cause conditioned taste aversion (CTA). ICV administration of alpha-MSH, however, did cause weak CTA. These results indicate that the potent effects on feeding of MC3-R and MC4-R agonists when injected into the PVN are not due to aversive effects.     

Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity

The paraventricular hypothalamic nucleus (PVN) appears to integrate orexigenic properties of a novel peptide, ghrelin. Thus, we examined central mechanisms underlying feeding generated by intra-PVN ghrelin. We established that 0.03 nmol of PVN-injected ghrelin was the lowest dose increasing food consumption and it induced c-Fos immunoreactivity (a marker of neuronal activation) in the PVN itself, as well as in other feeding-related brain areas, including the hypothalamic arcuate and dorsomedial nuclei, central nucleus of the amygdala, and nucleus of the solitary tract. We conclude that the PVN, as part of larger central circuitry, mediates orexigenic properties of ghrelin.     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

Alpha-melanocyte-stimulating hormone mediates melanin-concentrating hormone-induced anorexigenic action in goldfish

In goldfish, intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits feeding behavior, and fasting decreases hypothalamic MCH-like immunoreactivity. However, while MCH acts as an anorexigenic factor in goldfish, in rodents MCH has an orexigenic effect. Therefore, we examined the involvement of two anorexigenic neuropeptides, alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH), in the anorexigenic action of MCH in goldfish, using an alpha-MSH receptor antagonist, HS024, and a CRH receptor antagonist, alpha-helical CRH((9-41)). ICV injection of HS024, but not alpha-helical CRH((9-41)), suppressed MCH-induced anorexigenic action for a 60-min observation period. We then examined, using a real-time PCR method, whether MCH affects the levels of mRNAs encoding various orexigenic neuropeptides, including neuropeptide Y (NPY), orexin, ghrelin and Agouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for NPY and ghrelin, but not for orexin and AgRP. These results indicate that the anorexigenic action of MCH in the goldfish brain is mediated by the alpha-MSH signaling pathway and is accompanied by inhibition of NPY and ghrelin synthesis.     

Ghrelin is an orexigenic and metabolic signaling peptide in the arcuate and paraventricular nuclei

Ghrelin is a 28-amino acid acylated peptide and is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). The GHS-R is expressed in hypothalamic nuclei, including the arcuate nucleus (Arc) where it is colocalized with neuropeptide Y (NPY) neurons. In the present study, we examined the effects of ghrelin on feeding and energy substrate utilization (respiratory quotient; RQ) following direct injections into either the arcuate or the paraventricular nucleus (PVN) of the hypothalamus. Ghrelin was administered at the beginning of the dark cycle at doses of 15-60 pmol to male and female rats. In feeding studies, food intake was measured 2 and 4 h postinjection. Separate groups of rats were injected with ghrelin, and the RQ (VCO(2)/VO(2)) was measured using an open circuit calorimeter over a 4-h period. Both Arc and PVN injections of ghrelin increased food intake in male and female rats. Ghrelin also increased RQ, reflecting a shift in energy substrate utilization in favor of carbohydrate oxidation. Because these effects are similar to those observed after PVN injection of NPY, we then assessed the impact of coinjecting ghrelin with NPY into the PVN. When rats were pretreated with very low doses of ghrelin (2.5-10 pmol), NPY's (50 pmol) effects on eating and RQ were potentiated. Overall, these data are in agreement with evidence suggesting that ghrelin functions as a gut-brain endocrine hormone implicated in the regulation of food intake and energy metabolism. Our findings are also consistent with a possible interactive role of hypothalamic ghrelin and NPY systems.     

Impact of [d-Lys3]-GHRP-6 and feeding status on hypothalamic ghrelin-induced stress activation

Ghrelin administration directly into hypothalamic nuclei, including the arcuate nucleus (ArcN) and the paraventricular nucleus (PVN), alters the expression of stress-related behaviors. In the present study we investigated the effect of feeding status on the ability of ghrelin to induce stress and anxiogenesis. Adult male Sprague Dawley rats were implanted with guide cannula targeting either the ArcN or PVN. In the first experiment we confirmed that ArcN and PVN ghrelin treatment produced anxiety-like behavior as measured using the elevated plus maze (EPM) paradigm. Ghrelin was administered during the early dark cycle. Immediately after microinjections rats were placed in the EPM for 5 min. Both ArcN and PVN treatment reduced open arm exploration. The effect was attenuated by pretreatment with the ghrelin 1a receptor antagonist [d-Lys³]-GHRP-6. In a separate group of animals ghrelin was injected into either nucleus and rats were returned to their home cages for 60 min with free access to food. An additional group of rats was returned to home cages with no food access. After 60 min with or without food access all rats were tested in the EPM. Results indicated that food consumption just prior to EPM testing reversed the avoidance of the open arms of the EPM. In contrast, rats injected with ghrelin, placed in their home cage for 60 min without food, and subsequently tested in the EPM, exhibited an increased avoidance of the open arms, consistent with stress activation. Overall, our findings demonstrate that ghrelin 1a receptor blockade and feeding status appear to impact the ability of ArcN and PVN ghrelin to elicit stress and anxiety-like behaviors.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Melanocortin 4 Receptors in the Paraventricular Nucleus Modulate the Adipose Afferent Reflex in Rat

Background and Aim

Paraventricular nucleus (PVN) of hypothalamus is an important central component in modulating adipose afferent reflex (AAR). Melanocortin receptors (MC3/4Rs) expressions are found in the hypothalamic PVN. This study was designed to determine the roles of MC3/4Rs in the PVN in modulating the AAR and its downstream signaling pathway in normal rats.

Methodology/Principal Findings

Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in anaesthetized rats. AAR was evaluated using RSNA and MAP responses to capsaicin injection into the inguinal white adipose tissue (iWAT). Microinjection of the MC3/4R agonist melanotan II (MTII) into the PVN enhanced the AAR. The MC3/4R antagonist SHU9119 or MC4R antagonist HS024 attenuated the AAR and abolished MTII-induced AAR response. The adenylate cyclase (AC) inhibitor SQ22536 or the protein kinase A (PKA) inhibitor Rp-cAMP attenuated the AAR and the effect of MTII on the AAR was abolished by pretreatment with SQ22536 or Rp-cAMP in the PVN. Furthermore, both PVN microinjection of MTII and iWAT injection of capsaicin increased the cAMP level in the PVN. SHU9119 in the PVN abolished the increase in cAMP level which induced by iWAT injection of capsaicin.

Conclusion

The activation of MC4Rs rather than MC3Rs enhances the AAR, and a cAMP-PKA pathway is involved in the effects of MC4Rs in the PVN.     

beta-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R?

alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.4+/-0.4 nmol/l versus alpha-MSH, 324+/-16 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.0+/-0.4 nmol/l versus alpha-MSH, 22.5+/-2.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.     

Functional interaction between nociceptin/orphanin FQ and alpha-melanocyte-stimulating hormone in the regulation of feeding

Nociceptin/orphanin FQ (N/OFQ), an endogenous agonist of the opioid N/OFQ (NOP) receptor, increases food intake when administered centrally. As N/OFQ is part of a larger neural network that governs consummatory behavior, presumably its orexigenic properties stem from interplay with other neuropeptidergic components of the feeding-related circuitry. One such peptide may be the ligand of the melanocortin-3 and -4 receptors, alpha-melanocyte-stimulating hormone (alpha-MSH), which is known to inhibit food intake. The aim of the present study was to establish whether there is a functional "interaction" between N/OFQ and alpha-MSH in the regulation of feeding. By using double immunostaining for c-Fos and alpha-MSH, we found that intracerebroventricular (i.c.v.) injection of N/OFQ at a 10nmol dose that moderately prolongs deprivation-induced food intake in rats, decreases activation of alpha-MSH neurons involved in feeding termination. However, i.c.v. injections of alpha-MSH at doses previously established to reduce deprivation-induced feeding, do not decrease hyperphagia generated by N/OFQ in ad libitum-fed animals. Our results suggest that while alpha-MSH does not appear to modify the orexigenic response to N/OFQ in sated rats, the NOP receptor ligand promotes a decrease in activation of neurons synthesizing the anorexigenic peptide, alpha-MSH, at the time of re-feeding. Thus, to some degree, the stimulatory effect of N/OFQ on consumption may arise from this peptide's inhibitory influence on activity of anorexigenic pathways containing alpha-MSH.     

1H-NMR studies on a potent and selective antagonist at human melanocortin receptor 4 (hMC-4R)

Melanocortin receptor 4 (MC-4R) is involved in the regulation of energy balance and body weight, and recognizes alpha-, beta-, and gamma-melanocyte stimulating hormones (alpha-, beta-, gamma-MSH). In the search for compounds that regulate food intake and body weight, two synthetic lactam-derivative ligands of alpha-MSH were discovered, MTII and SHU9119. MTII is an agonist and reduces food intake in rats, whereas SHU9119 is an antagonist, and increases food intake and body weight in rats. MTII and SHU9119 are nonselective compounds to MC-4R. To enhance the potency and selectivity at the human MC-4R (hMC-4R), we recently synthesized analogs of SHU9119 (M. A. Bednarek, T. MacNeil, R. N. Kalyani, R. Tang, Van der L. H. T. Ploeg, and D. H. Weinberg, Journal of Medicinal Chemistry, 2001, Vol. 44, pp. 401-409), wherein compound 1 was the most selective for hMC-4R. Replacement of D-Nal by L-Nal in compound 1 made compound 2 weakly active. Comparison of the structures by NMR and molecular modeling of compounds 1 and 2 vs SHU9119 and MTII indicated that, even though they existed as an average of several conformations in solution, there were distinctions in their structures. The gamma-methylene protons of Arg in compound 1 were nonequivalent and shielded probably by the aromatic ring of Nal. The NHi-NHi+1 NOE cross peaks and the temperature coefficients of the amide protons around the "essential core" Nal/Phe7-Arg8-Trp9, required for high affinity and high selectivity at hMC-4R, were indicative of a possible turn structure for these compounds but with differences in their NOE strengths and temperature coefficient values. Molecular modeling of these compounds based on their NMR data showed that the essential core appeared as a "V" shape with two different orientations, one for compound 1 and some of the conformers of SHU9119 and MTII, and the other for compound 2 and some other conformers of SHU9119 and MTII. The remaining conformers of SHU9119 and MTII, which did not map to compound 1 or 2, suggested that they were outside of the hMC-4R binding envelop. These observations may lead to conjectures as to why compound 1 is highly active and selective toward hMC-4R.     

Centrally administered MTII affects feeding, drinking, temperature, and activity in the Sprague-Dawley rat.

MTII, an agonist of melanocortinergic receptors, is a well-documented anorexigenic agent in rats. Many investigators have reported its effects on feeding without considering concurrent alterations in other behaviors. Accordingly, we performed studies to simultaneously measure nocturnal feeding, drinking, activity, and temperature of rats after intracerebroventricular (third ventricle) administration of a wide dose range of MTII (0.05-500 ng). We observed that MTII modulates these physiological parameters in a dose-dependent manner. Low doses of MTII (0.05 ng) caused reductions in feeding without alterations in body temperature, drinking, or activity. In contrast, hyperthermia and disrupted drinking patterns, along with food intake reductions, were evident at doses exceeding 50 ng. The fact that low doses altered only feeding, whereas higher doses affected a range of parameters, suggests that certain melanocortin-induced behavioral changes may be mediated by distinct populations of melanocortin receptors with varying affinities or that those changes seen at higher doses may be nonspecific in nature.     

Effect of i.c.v. infusion of the alpha-MSH agonist MTII on meal patterns in male rats following nicotine withdrawal

The present study explored the role of endogenous alpha-MSH in the alteration of meal patterns induced by nicotine (NIC) withdrawal. Male Sprague Dawley rats bearing third ventricle cannulas were placed in computerized food intake monitors. On days 1-21, the rats were given 4 mg/kg/day of NIC or saline (SAL) in four equal i.p. doses during the dark period. NIC suppressed (P < 0.05) food intake only during the first week. The normalization of food intake occurred when the reduced meal size of the NIC injected rats was countered by an increase in meal number. Despite the normalization of 24-h food intake, body weight in NIC rats was decreased (P < 0.05) for 21 days. On day 22, the rats were divided into 4 groups (n's = 7-8 each) and injected into the third ventricle with various doses of the alpha-MSH agonist MTII or artificial cerebrospinal fluid (aCSF): SAL + aCSF, SAL + MTII, NIC + aCSF, NIC + MTII. Infusion of MTII (30 ng/rat) suppressed (P < 0.01) dark phase food intake in both groups, but the NIC + MTII group ate (P < 0.05) more than the SAL + MTII group. Meal number during the dark phase was suppressed by MTII, but the NIC + MTII group took significantly more meals that the SAL + MTII group. Infusion of MTII suppressed meal size in SAL and NIC treated rats, but this effect was attenuated in NIC treated rats. All meal parameters normalized by the day after i.c.v. infusion. These data indicate that NIC treatment differentially affects the neural controls of meal number and meal size and attenuates the suppression by MTII of meal number and meal size.     

Parathyroid hormone-related protein and lung injury after pulmonary thromboendarterectomy

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of alpha-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC(50) values for melanocortin agonists (alpha-MSH, NDP-MSH, and MTII) and a pA2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.     

Activation of the melanocortin-4 receptor causes enhanced excitation in presympathetic paraventricular neurons in obese Zucker rats

Sympathetic nerve activity is increased in obesity-related hypertension. However, the central mechanisms involved in the increased sympathetic outflow remain unclear. The hypothalamic melanocortin system is important for regulating energy balance and sympathetic outflow. To understand the mechanisms by which the melanocortin systems regulates sympathetic outflow, we investigated the role of melanocortin 4 receptors (MC4R) in regulating presympathetic paraventricular nucleus (PVN) neurons. We performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the rostral ventrolateral medulla in brain slices from obese zucker rats (OZRs) and lean zucker rats (LZRs). The MC4R agonists melanotan II (MTII) and α-melanocyte-stimulating hormone (α-MSH) increased the firing activity and depolarized the labeled PVN neurons from both LZRs and OZRs in a concentration-dependent manner. MTII produced significant greater increase in the firing activity in OZRs than in LZRs. Blocking MC4R with the specific antagonist SHU9119 had no effect on the basal firing rate but abolished the MTII-induced increase in the firing rate in both OZRs and LZRs. Furthermore, intracellular dialysis of guanosine 5'-O-(2-thodiphosphate), but not bath application of kynurenic acid and bicuculline, eliminated the MTII-induced increase in firing activity. In addition, MTII had no effect on the frequency and amplitude of glutamatergic excitatory postsynaptic currents and GABAergic inhibitory postsynaptic currents in labeled PVN neurons. Collectively, our findings suggest that MC4R contributes to the elevated excitability of PVN presympathetic neurons, which may be involved in obesity-related hypertension.     

Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake

Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor tyrosine kinase B (TrkB) are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF in the hypothalamic paraventricular nucleus (PVN) on feeding. BDNF injected unilaterally or bilaterally into the PVN of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and 24- to 48-h postinjection intervals. Effective doses producing inhibition of feeding behavior did not establish a conditioned taste aversion. PVN BDNF significantly decreased PVN neuropeptide Y (NPY)-induced feeding at 1, 2, and 4 h following injection. BDNF administration in the PVN abolished food-restriction-induced NPY gene expression in the hypothalamic arcuate nucleus. In conclusion, BDNF in the PVN significantly decreases food intake and body weight gain, suggesting that the PVN is an important site of action for BDNF in its effects on energy metabolism. Furthermore, BDNF appears to interact with NPY in its anorectic actions, although a direct effect on NPY remains to be established.     

Corticotropin-releasing hormone mediates alpha-melanocyte-stimulating hormone-induced anorexigenic action in goldfish

alpha-Melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH) both suppress food intake, and the alpha-MSH- or CRH-signaling pathway has possible potency to mediate anorexigenic actions induced by most other neuropeptides in goldfish. Therefore, using specific receptor antagonists, we examined whether the anorexigenic actions of alpha-MSH and CRH mutually interact. The inhibitory effect of ICV injection of the alpha-MSH agonist, melanotan II (MT II), on food intake was abolished by treatment with a CRH 1/2 receptor antagonist, alpha-helical CRH((9-41)), whereas the anorexigenic action of ICV-injected CRH was not affected by treatment with a melanocortin 4 receptor antagonist, HS024. This led us to investigate whether alpha-MSH-containing neurons in the goldfish brain have direct inputs to CRH-containing neurons, using confocal laser scanning microscopy. alpha-MSH- and CRH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. alpha-MSH-containing nerve fibers or endings lay in close apposition to CRH-containing neurons in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These results indicate that, in goldfish, alpha-MSH-induced anorexigenic action is mediated by the CRH-signaling pathway, and that CRH plays a crucial role in the regulation of feeding behavior as an integrated anorexigenic neuropeptide in this species.     

Differential feeding responses to central alpha-melanocyte stimulating hormone in genetically low and high body weight selected lines of chickens

This study was conducted to compare the effects of central alpha-MSH, a potent anorexigenic signal, in lines of chickens that have undergone long-term divergent selection for low (LWS) or high (HWS) body weight. Chicks from both lines were centrally injected with 0, 24, 120 or 600 pmol alpha-MSH and feed and water intake were concurrently measured thereafter for a total of 180 min. The LWS line responded to all doses of alpha-MSH with a similar potent decrease in feed intake at all observation times. The HWS line only responded to 600 pmol alpha-MSH with decreased feed intake. alpha-MSH did not influence water intake in either line. To determine if differential hypothalamic signaling was associated with the anorexigenic effect, c-Fos immunoreactivity was measured in appetite-related hypothalamic nuclei after 600 pmol central alpha-MSH injections. c-Fos immunoreactivity was increased in the dorsomedial hypothalamus, paraventricular nucleus (PVN) and ventromedial hypothalamus in both lines after alpha-MSH; however, the magnitude of increase was greater in LWS than in HWS chicks at the PVN (136% vs. 47% increase over controls, respectively). Based on behavior observations, the number of feeding and exploratory pecks is decreased with greater magnitude after alpha-MSH in the LWS line. Additionally, alpha-MSH was associated with increased deep rest in both lines, and may be a secondary effect to reduced ingestion. These data support that the LWS line has a lower threshold for the anorexigenic effect of central alpha-MSH while in the HWS line this threshold is higher, and that this difference may be associated with differential hypothalamic signaling. Genetic variation exists in the threshold of anorexigenic response for central alpha-MSH in LWS and HWS lines of chickens with possible implications to other species including humans.