Regulation of the mesocorticolimbic and mesostriatal dopamine systems by α-melanocyte stimulating hormone and agouti-related protein

The melanocortin system of the hypothalamus, including the neuropeptides α-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP), and their receptors, the melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R), have been well-studied for their roles in the central control of feeding and body weight. In this review, we discuss the evidence demonstrating that αMSH and AgRP also act on the mesocorticolimbic and mesostriatal dopamine systems to regulate a wide variety of behaviors. In addition to the well described ability of αMSH to increase dopamine transmission and to increase grooming and rearing when injected directly into the ventral tegmental area, a growing body of evidence indicates that αMSH and AgRP can also act on dopamine pathways to regulate feeding and drug abuse, including reward-related behaviors toward food and drugs. Increasing our understanding of how αMSH and AgRP act on dopamine pathways to affect behavior may allow for identification of new strategies to combat disorders involving dysfunction of dopamine pathways, such as obesity and drug abuse.     

Orexin expression is regulated by alpha-melanocyte-stimulating hormone

The hypothalamic melanocortin system plays a fundamental role in the regulation of energy homeostasis. Orexins (hypocretins) are also involved in a diverse range of physiological processes, including food intake. Previous evidence has suggested that hypothalamic orexin expression may be influenced by the central melanocortin system. Here, we studied orexin mRNA levels in pro-opiomelanocortin-deficient (Pomc(-/-)) mice, a mouse model lacking all endogenously produced melanocortin peptides. Orexin expression in the lateral hypothalamus was significantly increased in corticosterone deficient Pomc(-/-) mice. Furthermore, when circulating glucocorticoids were restored to levels within the physiological range, orexin expression remained elevated. However, i.c.v. administration of the melanocortin alpha-melanocyte-stimulating hormone (MSH) to Pomc(-/-) mice reduced orexin expression back down to wild-type levels. This was independent of the effects of alpha-MSH on food intake because elevated orexin expression persisted in Pomc(-/-) mice pairfed to alpha-MSH-treated animals. These data indicate that alpha-MSH may play a role in the regulation of orexin expression in Pomc(-/-), with an elevation in orexin levels contributing to the hyperphagia seen in these animals.     

Agouti-related protein prevents self-starvation

Food restriction leads to a paradoxical increase in physical activity and further suppression of food intake, such as observed in anorexia nervosa.(1,2) To understand this pathophysiological process, we induced physical hyperactivity and self-starvation in rats by restricting food in the presence of running wheels. Normally, decreased melanocortin receptor activity will prevent starvation.(3,4) However, we found that self-starvation increased melanocortin receptors in the ventral medial hypothalamus, a brain region involved in eating behavior.(5) Suppression of melanocortin receptor activity, via central infusion of Agouti-related protein (AgRP), increased survival rate in these rats by counteracting physical hyperactivity, food intake suppression as well as deregulated body temperature. We conclude that self-starvation may result from insufficient suppression of central melanocortin receptor activity.     

Effects of fluoxetine administration on hypothalamic melanocortin system in obese Zucker rats

The aim of the present work was to study the potential involvement of melanocortin system in the anorectic mechanism of fluoxetine, a selective serotonin reuptake inhibitors, in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. RT-PCR for pro-opiomelanocortin (POMC), Agouti gene related peptide (AgRP) and melanocortin receptor 4 (MC4-R) in the hypothalamus, as well as regional immunostaining for alpha-melanocyte stimulating hormone (alpha-MSH) and MC4-R were carried out. Fluoxetine administration increased POMC expression and reduced MC4-R expression in the hypothalamus, without changes in AgRP mRNA levels. Moreover, an increase in the numbers of alpha-MSH positively immunostained neural cells in the hypothalamic arcuate nucleus (ARC), as well as a significant decrease in the numbers of neural cells positively immunostained for MC4-R in the paraventricular nucleus (PVN), without changes in lateral hypothalamic area (LHA), were observed. These results suggest the involvement of alpha-MSH in central fluoxetine anorectic action.     

Evidence for a gut-brain axis used by glucagon-like peptide-1 to elicit hyperglycaemia in fish

In mammals, glucagon-like peptide-1 (GLP-1) produces changes in glucose and energy homeostasis through a gut-pancreas-brain axis. In fish, the effects of GLP-1 are opposed to those described in other vertebrates, such as stimulation of hyperglycaemia and the lack of an effect of incretin. In the present study conducted in a teleost fish such as the rainbow trout, we present evidence of a gut-brain axis used by GLP-1 to exert its actions on glucose and energy homeostasis. We have assessed the effects of GLP-1 on glucose metabolism in the liver as well as the glucose-sensing potential in the hypothalamus and hindbrain. We confirm that peripheral GLP-1 administration elicits sustained hyperglycaemia, whereas, for the first time in a vertebrate species, we report that central GLP-1 treatment increases plasma glucose levels. We have observed (using capsaicin) that at least part of the action of GLP-1 on glucose homeostasis was mediated by vagal and splanchnic afferents. GLP-1 has a direct effect in parameters involved in glucose sensing in the hindbrain, whereas, in the hypothalamus, changes occurred indirectly through hyperglycaemia. Moreover, in the hindbrain, GLP-1 altered the expression of peptides involved in the control of food intake. We have elaborated a model for the actions of GLP-1 in fish in which this peptide uses a mammalian-like ancestral gut-brain axis to elicit the regulation of glucose homeostasis in different manner than the model described in mammals. Finally, it is worth noting that the hyperglycaemia induced by this peptide and the lack of incretin function could be related to the glucose intolerance observed in carnivorous teleost fish species such as the rainbow trout.     

Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues

Central administration of neuropeptide Y (NPY) potently induces feeding and its abundance in the hypothalamus increases when energy stores fall. Consequently, NPY is considered to be a physiological effector of feeding behavior. Surprisingly, NPY-deficient (NPY-/-) mice feed and grow normally with ad libitum access to food and manifest a normal hyperphagic response after fasting, suggesting that other feeding effectors may compensate for the lack of NPY. Agouti-related protein (AgRP), a melanocortin receptor antagonist, can also stimulate feeding behavior when administered centrally and is coexpressed in a majority of hypothalmamic NPY-ergic neurons, making AgRP a candidate compensatory factor. To test this possibility, we evaluated AgRP mRNA and protein expression, as well as responsiveness to centrally administered AgRP in NPY-/- mice. These studies demonstrate that hypothalamic AgRP mRNA and immunoreactivity are upregulated with fasting and that these increases are not affected by NPY deficiency. Interestingly, NPY-/- mice are hypersensitive to central administration of AgRP(83-132), yet exhibit a normal response to centrally administered MTII, a melanocortin receptor agonist. These data suggest that if AgRP compensates for the lack of NPY in NPY-/- mice, it is not at the level of AgRP synthesis and may instead involve alterations in the postsynaptic signaling efficacy of AgRP. Moreover, the effects of AgRP are not limited to its actions at the melanocortin-4 receptor (MC4R), because MC4R-deficient (MC4R-/-) mice manifest a significant response to centrally administered AgRP. These data imply that AgRP has additional targets in the hypothalamus.     

Interleukin-6 regulates the expression of hypothalamic neuropeptides involved in body weight in a gender-dependent way

Interleukin (IL)-6 has been involved in the control of body weight and body fat. Nevertheless, the mechanisms underlying these effects are not completely understood because central and peripheral actions of IL-6 are plausible. To gain further insight into the central effects of IL-6, we used transgenic mice expressing the IL-6 gene under the control of the glial fibrillary acidic protein (GFAP) promoter (GFAP-IL-6 mice), therefore with central nervous system-restricted over-expression of IL-6, and we studied the expression of the main neuropeptides responsible for energy homeostasis in specific hypothalamic nuclei. Neuropeptide Y (NPY), agouti-related peptide (AgRP), melanin-concentrating hormone (MCH), prepro-orexin (preproOX) (orexigenic and anabolic neuropeptides), pro-opiomelanocortin (POMC) and corticotrophin-releasing hormone (CRH) (anorexigenic and catabolic peptides) mRNA levels were determined using in situ hybridisation in young (2-4 month-old) and old (10-12 month-old) female and male mice under different feeding conditions: normal diet (control) and high-fat diet (HFD), and 24 h-food deprivation. In GFAP-IL-6 females fed a control diet (GFAP-IL-6-control), we showed a significant decrease in NPY and AgRP mRNA levels at all ages, and a late increase in POMC expression (only significant in older animals). These differences were blunted in HFD mice. By contrast, GFAP-IL-6-control males showed a decrease in CRH mRNA content at early ages (2-4 months), and an increase in older mice (10-12 months). Interestingly, these differences were again blunted in HFD mice. Finally, central IL-6 was not able to counteract the effects of 24 h of fasting on body weight, plasma glucose levels and the mRNA content of the peptides evaluated in the present study. Our results demonstrate that IL-6 may regulate the expression of hypothalamic neuropeptides involved in the control of body weight and body fat acting at the central level in a gender- and age-dependent way.     

Effects of melanocortin receptor ligands on thyrotropin-releasing hormone release: evidence for the differential roles of melanocortin 3 and 4 receptors

The hypothalamic melanocortin system is important in the central regulation of food intake and body weight. We have previously demonstrated that intracerebroventricular administration of alpha-melanocyte stimulating hormone (alpha-MSH), a nonselective MC3 and MC4 receptor agonist, stimulated plasma thyroid-stimulating hormone, and agouti-related protein (AgRP), an MC3 and MC4 receptor antagonist, suppressed it. In this study, we investigated the effects of MC3 and MC4 receptor (MC3-R and MC4-R) selective agonists and antagonists on the release of thyrotropin-releasing hormone (TRH) from hypothalamic explants in vitro. alpha-MSH stimulated TRH release from the rat hypothalamic explants (alpha-MSH 100 nm 230 +/- 22.9% basal, P < 0.005). In contrast, gamma 2-MSH, a selective MC3-R agonist, suppressed TRH release (gamma 2-MSH 10 microns 76.2 +/- 7.4% basal, P < 0.05). AgRP (83-132), a nonselective MC3/4-R antagonist, induced no change in TRH release whilst JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH 11-22), a selective MC4-R antagonist, suppressed it (JKC-363 10 nm 57.2 +/- 11.5% basal, P < 0.05). Both AgRP (83-132) and JKC-363 blocked alpha-MSH stimulated TRH release but only AgRP (83-132) blocked the inhibitory effect of gamma 2-MSH on TRH release. These data suggest differential roles for the MC3 and MC4 receptors in TRH release; MC3-R agonism inhibiting and MC4-R agonism stimulating TRH release.     

Role of alpha-MSH and related peptides in the central nervous system

Alpha-melanocyte stimulating hormone (alpha-MSH) is a tridecapeptide secreted by intermediate lobe cells and synthesized in the brain as well. As a hormonal peptide, the physiological function of alpha-MSH consists mainly in the control of pigment movements within dermal melanophores. At the pituitary level, alpha-MSH secretion is under multifactorial control: it is inhibited by dopamine and GABA and stimulated by corticoliberin (CRF), thyroliberin (TRH), beta-adrenergic agonists and (or) serotonin. Identification of alpha-MSH containing neurons in the hypothalamus and other brain regions (septum, thalamus, mid-brain, striatum, hippocampus, cerebral cortex and spinal cord) has been carried out by means of immunological and biochemical techniques combined with bioassays. In the central nervous system (CNS) as in the hypophysis, alpha-MSH is synthesized from a high molecular weight precursor, pro-opiomelanocortin (POMC). Maturation of this protein yield similar end products in the hypothalamus and the intermediate lobe. Several peptides chemically related to alpha-MSH are generated including the desacetyl and monoacetyl (authentic alpha-MSH) forms; the latter has the greatest behavioral activity. The demonstration that alpha-MSH has numerous central nervous system effects has raised the possibility that this neuropeptide acts as a neuromodulator or a neurotransmitter. In the rat, intra-cerebroventricular administration of ACTH/MSH peptides induces the stretching-yawning syndrome (SYS) which is frequently preceded by excessive grooming. This excessive grooming is blocked by neuroleptics indicating that the central dopaminergic neurons are implicated in this behavioral effect of the peptide. alpha-MSH is involved in memory, arousal and attention; in hypophysectomized animals, the learning ability is restored after administration of MSH or related peptides. Injection of alpha-MSH delays also extinction of passive avoidance behavior and affects performances motivated by hunger as well as aggressive behavior. Recent studies concerning the role of alpha-MSH have been undertaken in human beings. The effects of MSH-related peptides favour a role of these peptides in arousal: they maintain a high level of vigilance and improve visual discrimination. These behavioral changes were accompanied by marked changes in CNS electrophysiology. Current studies, which aim at establishing a neurotransmitter function for alpha-MSH, concern the distribution and characterization of alpha-MSH receptors in the central nervous system and the mechanism controlling the release of neuronal alpha-MSH.     

Plasma agouti-related protein levels in women with anorexia nervosa

Agouti-related protein (AGRP) is the competitive antagonist of alpha-melanocyte stimulating hormone (alpha-MSH) located at melanocortin receptors 3 and 4 (MC3R and MC4R), and also acts as an MC4R inverse agonist. Hypothalamic AGRP controls food intake and body weight in rodents. It has also been found in human plasma. To study the possibility of disturbances in melanocortin receptor-related peptides in eating disorders, plasma AGRP, alpha-MSH, and leptin levels were measured in 18 female patients with anorexia nervosa (AN) (age, 23.5+/-7.1 yr; body mass index (BMI) 14.5+/-1.8 kg/m(2)) and 17 age-matched female controls (age, 25.8+/-3.9 yr; BMI 20.2+/-1.6 kg/m(2)). Blood samples were collected after overnight fasting, and plasma peptides levels were measured using ELISA. Plasma AGRP levels increased significantly in AN patients when compared with controls (P<0.01) while plasma alpha-MSH levels were not significantly different. Plasma leptin levels decreased significantly in AN patients when compared with controls (P<0.001). In addition, plasma AGRP levels were negatively correlated with leptin (r=-0.41, P<0.01) and BMI (r=-0.40, P<0.05) in all subjects. In conclusion, plasma AGRP elevation may be related to energy homeostasis disturbance in AN, and in addition to leptin, peripheral AGRP levels could be used as a nutritional marker in AN patients.     

The MC4 receptor mediates alpha-MSH induced release of nucleus accumbens dopamine.

Strong evidence suggests a functional link between the melanocortin and dopamine systems. alpha-Melanocyte stimulating hormone (alpha-MSH) induced grooming behaviour, which can be blocked by dopamine receptor antagonists, is associated with increased dopaminergic transmission in the striatal regions. Whether this effect is mediated specifically by melanocortin (MC) receptors has not previously been established. Using in vivo microdialysis on anesthesized rats we have shown that alpha- MSH administered into the ventral tegmental area induced a significant increase in dopamine and DOPAC levels in the nucleus accumbens. This increase was completely blocked by pre-treatment with the MC4 receptor selective antagonist HS131, indicating that the effects of alpha-MSH on dopamine transmission may be mediated by the MC4 receptor.     

Distribution of corticotropin-releasing hormone in the developing zebrafish brain

Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh-expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain.     

Melanocortin receptors as drug targets for disorders of energy balance

There is overwhelming evidence that the brain melanocortin system is a key regulator of energy balance, and dysregulations in the brain melanocortin system can lead to obesity. The melanocortin system is one of the major downstream leptin signaling pathways in the brain. In contrast to leptin, preclinical studies indicate that diet-induced obese animals are still responsive to the anorectic effects of melanocortin receptor agonists, suggesting the melanocortin system is an interesting therapeutic opportunity. Besides regulating energy balance, melanocortins are involved in a variety of other neuroendocrine processes, including inflammation, blood pressure regulation, addictive and sexual behavior, and sensation of pain. This review evaluates the melanocortin system function from the perspective to use specific melanocortin (MC) receptors as drug targets, with a focus on the treatment of obesity and eating disorders in humans, and the implications this may have on mechanisms beyond the control of energy balance.     

Short‐Days Induce Weight Loss in Siberian Hamsters Despite Overexpression of the Agouti‐Related Peptide Gene

Many vertebrates express profound annual cycles of body fattening, although it is not clear whether these represent differential activity of the central pathways known to mediate homeostatic control of food intake and energy expenditure, or whether the recent discovery of a major role for pars tuberalis‐ependymal signalling points towards novel mechanisms. We examined this in the Siberian hamster (Phodopus sungorus) by using gene transfection to up‐regulate a major orexigenic peptide, agouti‐related peptide (AgRP), and then determined whether this increased anabolic drive could prevent the short‐day induced winter catabolic state. Infusions of a recombinant adeno‐associated virus encoding an AgRP construct into the hypothalamus of hamsters in the long‐day obese phase of their seasonal cycle produced a 20% gain in body weight over 6 weeks compared to hamsters receiving a control reporter construct, reflecting a significant increase in food intake and a significant decrease in energy expenditure. However, all hamsters showed a significant, prolonged decrease in body weight when exposed to short photoperiods, despite the hamsters expressing the AgRP construct maintaining a higher food intake and lower energy expenditure relative to the control hamsters. Visualisation of the green fluorescent protein reporter and analysis of AgRP‐immunoreactivity confirmed widespread expression of the construct in the hypothalamus, which was maintained for the 21‐week duration of the study. In conclusion, the over‐expression of AgRP in the hypothalamus produced a profoundly obese state but did not block the seasonal catabolic response, suggesting a separation of rheostatic mechanisms in seasonality from those maintaining homeostasis of energy metabolism.     

The 36K protein of zebrafish CNS myelin is a short-chain dehydrogenase

Previous studies identified homologues to mammalian myelin genes expressed in the teleost central nervous system (CNS), including myelin basic protein (MBP), protein zero (P0), and a member of the proteolipid protein family, DM20. In addition, an uncharacterized 36-kDa (36K) protein is a major component of teleost myelin, but is not a major component of myelin in other species. In the present study, we sought to better understand myelin proteins and myelination in one teleost, zebrafish, by molecular characterization of the zebrafish 36K protein. Purified zebrafish CNS myelin was isolated and the amino acid sequences of peptides present in the 36-kDa band were determined by mass spectrometry. These sequences matched a previously uncharacterized EST in The Institute for Genome Research (TIGR) zebrafish database that is related to the short-chain dehydrogenase/reductase (SDR) protein family. In vitro expression of the zebrafish 36K cDNA in Neuro 2a cells resulted in a protein product that was recognized by a 36K polyclonal antibody. The zebrafish 36K mRNA and protein expression patterns were determined and correlated to other known myelin gene expression profiles. In addition, we determined by in situ hybridization that a human 36K homologue (FLJ13639) is expressed in oligodendrocytes and neurons in the adult human cortex. This study identified a major myelin protein in zebrafish, 36K, as a member of the SDR superfamily; an expression pattern similar to other myelin genes was demonstrated.     

Roles of the melanocortin-4 receptor in antipyretic and hyperthermic actions of centrally administered alpha-MSH

Activation of central melanocortin receptors (MCR) inhibits fever but can also stimulate thermogenesis, and the mechanisms involved are unknown. To determine whether the long-recognized antipyretic effect of exogenous alpha-MSH is mediated by the melanocortin-4 receptor (MC4R), and what thermoeffector systems are involved, we tested the effects of intracerebroventricular (i.c.v.) injection of alpha-MSH on lipopolysaccharide (LPS, 30 microg/kg i.p.)-induced fever in rats, in the presence and absence of the selective MC4R antagonist HS014. Treatment with alpha-MSH (1 microg, i.c.v.) suppressed LPS-induced increases in core body temperature (Tc), whereas a lower dose (300 ng) was ineffective. Nevertheless, both alpha-MSH doses effectively inhibited LPS-induced peripheral vasoconstriction, the principal heat-conserving thermoeffector, as determined by changes in tail skin temperature (Tsk). This implies that the net antipyretic effect of alpha-MSH cannot be accounted for solely by modulation of heat loss effectors, but also involves other mechanisms. Surprisingly, central MC4-R blockade by coinjected HS014 (1 microg) not only prevented, but reversed the effect of alpha-MSH (1 microg) on Tc, thus resulting in augmented LPS-induced fever. In afebrile rats, alpha-MSH infusion caused a modest transient increase in Tc that was blocked by coinjected HS014, but was not accompanied by altered Tsk. Overall, the results support the hypothesis that the MC4R mediates the antipyretic effects of alpha-MSH. Paradoxically, in the presence of pharmacological MC4-R blockade during fever, exogenous alpha-MSH can exacerbate fever, probably by acting via other central MCR subtype(s). In normal animals, centrally injected alpha-MSH exerts a hyperthermic effect that is mediated by the MC4R, consistent with recent evidence that MC4R activation promotes energy expenditure in normal states through stimulation of thermogenesis.     

Evidence of interactions between melanocortin and opioid systems in regulation of feeding.

The aim of our experiments was to study the presumed functional relationship between the melanocortin and opioid systems in the regulation of food intake. We determined that a non-selective opioid receptor antagonist, naltrexone, at relatively low doses, decreases food intake induced by i.c.v. agouti-related protein (Agrp). We also observed that peripheral injection of naltrexone at a dose known to produce anorexigenic responses induced c-Fos immunoreactivity in significantly more arcuate nucleus alpha-MSH neurons than observed in control animals. The results of our study support the notion that the melanocortin and opioid systems interact in the regulation of food intake. Based on these data we speculate that opioid peptides suppress alpha-MSH-dependent satiety mechanisms; conversely, it is possible that the orexigenic action of Agrp is mediated via opioid dependent circuitry.     

Coexistence of melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the preoptic nucleus of the frog brain

Coexistence of MCH- and alpha-MSH-like peptides in specific neurons of the frog hypothalamus has been investigated on serial frozen sections using the indirect immunofluorescence method. In the anterior region of the preoptic nucleus, perikarya containing MCH- and alpha-MSH-immunoreactive materials were co-distributed and the two peptides were generally co-sequestered within the same neurons. In contrast, alpha-MSH immunoreactive neurons of the ventral infundibular nucleus did not contain any MCH-like peptide. These data suggest that MCH and alpha-MSH are transported by the same nerve fibers originating from preoptic perikarya and are likely released together by axon terminals. Since MCH and alpha-MSH exert antagonistic hormonal activities on dermal melanophores, our results suggest that the two regulatory peptides may also interact in the central nervous system.