Agouti-related protein (AgRP) and agouti signaling protein (ASIP) are homologs that play critical roles in energy balance and pigmentation, respectively, by functioning as antagonistic ligands at their cognate melanocortin receptors. Signaling specificity is mediated in part through receptor binding selectivity brought about by alterations in the cysteine-rich carboxy-terminal domains of the ligands. AgRP binds with high affinity to the melanocortin 3 receptor and the melanocortin 4 receptor, but not to the melanocortin 1 receptor (MC1R), whereas ASIP binds with high affinity to all three receptors. This work explores the structural basis for receptor selectivity by studying chimeric proteins developed by interchanging loops between the cysteine-rich domain of ASIP and the cysteine-rich domain of AgRP. Binding data demonstrate that melanocortin 4 receptor responds to all chimeras and is therefore highly tolerant of gross loop changes. By contrast, MC1R responds primarily to those chimeras with a sequence close to that of wild-type ASIP. Further analysis of binding and functional data suggests that the ASIP C-terminal loop (a six-amino-acid segment closed by the final disulfide bond) is essential for high-affinity MC1R binding and inverse agonism. Comparison with previously published molecular models suggests that this loop makes contact with the first extracellular loop of MC1R through a series of key hydrophobic interactions.

In mice and humans, binding of alpha-melanocyte--stimulating hormone to the melanocyte-stimulating--hormone receptor (MSHR), the protein product of melanocortin-1 receptor (MC1R) gene, leads to the synthesis of eumelanin. In the mouse, ligation of MSHR by agouti signaling protein (ASP) results in the production of pheomelanin. The role of ASP in humans is unclear. We sought to characterize the agouti signaling protein gene (ASIP) in a group of white subjects, to assess whether ASIP was a determinant of human pigmentation and whether this gene may be associated with increased melanoma risk. We found no evidence of coding-region sequence variation in ASIP, but detected a g.8818A-->G polymorphism in the 3' untranslated region. We genotyped 746 participants in a study of melanoma susceptibility for g.8818A-->G, by means of polymerase chain reaction and restriction fragment--length polymorphism analysis. Among the 147 healthy controls, the frequency of the G allele was.12. Carriage of the G allele was significantly associated with dark hair (odds ratio 1.8; 95% confidence interval [CI] 1.2--2.8) and brown eyes (odds ratio 1.9; 95% CI 1.3--2.8) after adjusting for age, gender, and disease status. ASIP g.8818A-->G was not associated independently with disease status. This is the first report of an association of ASIP with specific human pigmentation characteristics. It remains to be investigated whether the interaction of MC1R and ASIP can enhance prediction of human pigmentation and melanoma risk.

Here we report a heterozygous tandem duplication at the ASIP (agouti signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female patient with extreme childhood obesity. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase promoter, driving the generation of ASIP in patient-derived native and induced pluripotent stem cells for all germ layers and hypothalamic-like neurons. The patient's phenotype of early-onset obesity, overgrowth, red hair and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog nonagouti. ASIP represses melanocyte-stimulating hormone-mediated activation as a melanocortin receptor antagonist, which might affect eating behavior, energy expenditure, adipocyte differentiation and pigmentation, as observed in the index patient. As the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig Childhood Obesity cohort of 1,745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity.

Although the European roe deer (Capreolus capreolus) population of North-West Germany has a remarkable number of melanistic specimens between 10% and 25%, the underlying genetic mutation-causing melanism is still unknown. We used a gene targeting approach focusing on MC1R and ASIP as important genes of coat coloration. Overall, 1384 bp of MC1R and 2039 bp of ASIP were sequenced in 24 specimens and several SNPs were detected. But only the ASIP-SNP c.33G>T completely segregated both phenotypes leading to the amino acid substitution p.Leu11Phe. The SNP was further evaluated in additional 471 samples. Generally, all black specimens (n = 33) were homozygous TT, whereas chestnut individuals were either homozygote GG (n = 436) or heterozygote GT (n = 26). Considering the fact that all melanistic animals shared two mutated alleles of the strongly associated SNP, we concluded that melanism is inherited in a recessive mode in European roe deer.

Transcriptome analyses of bovine muscle tissue differing in intramuscular fat (IMF) content identified agouti signaling protein (ASIP) as a promising candidate gene for fat deposition. The protein is secreted from adipocytes and may serve as a signaling molecule in cross-talk between adipocytes and muscle fibers or other cells. Known receptors for ASIP are the melanocortin receptors (e.g., MC4R) and attractin (ATRN). The present study was conducted to determine relationships between the expression of ASIP and its receptors in different bovine tissues with fat deposition. Adipose tissues, liver, and longissimus muscle tissue were collected from 246 F2-generation bulls (Charolais × Holstein cross) and gene expression was measured with RT-qPCR. During analysis of subcutaneous fat (SCF) of all bulls, 17 animals were identified with a transposon-derived transcript (Exon2C) inserted in the ASIP gene and dramatically increased ASIP mRNA levels. Significant correlations between normalized mRNA values of SCF and phenotypic traits related to fat deposition were found in bulls without Exon2C. Three retrospectively assigned groups [Exon2C, n = 17; high carcass fat (HCF), n = 20; low carcass fat (LCF), n = 20] were further analyzed to verify expression differences and elucidate molecular reasons. Expression of ASIP could be detected in isolated muscle fibers and adipocytes of Exon2C bulls in contrast to HCF and LCF bulls, indicating ectopic ASIP expression if the transposon is present. Among adipose tissues, highest ASIP mRNA levels were measured in SCF with significantly higher values in HCF compared to LCF bulls (1.6-fold, P < 0.05). However, the protein abundance was below the detection limit in all bulls. Potential ASIP receptors were detected in most investigated tissues. The expression of MC4R was higher and of ATRN was lower in several tissues of LCF compared to HCF bulls, whereas MC1R was not differentially expressed. Bulls of the Exon2C group had lower ATRN mRNA values than HCF and LCF bulls in perirenal fat (PF), but higher (P < 0.05) values in muscle. Receptors were also expressed in tissues where ASIP mRNA was not detected. Consequently, those tissues could be targets for ASIP if it circulates.

Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP).

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, alpha-melanocyte-stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan-a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome-like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time- and dose-dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase-related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

The agouti locus encodes the agouti signalling protein (ASIP) which is involved in determining the switch from eumelanin to pheomelanin synthesis in melanocytes. In the domestic rabbit (Oryctolagus cuniculus) early studies indicated three alleles at this locus: A, light-bellied agouti (wild type); a(t), black and tan; a, black nonagouti. We characterized the rabbit ASIP gene and identified the causative mutation (an insertion in exon 2) of the black nonagouti allele whose frequency was evaluated in 31 breeds. Phylogenetic analysis of ASIP sequences from Oryctolagus and 9 other species of the family Leporidae placed Oryctolagus as sister species to Pentalagus and Bunolagus. Transcription analysis in wild type agouti rabbits revealed the presence of two major transcripts with different 5'-untranslated regions having ventral or dorsal skin specific expression. ASIP gene transcripts were also detected in all examined rabbit tissues distinguishing the rabbit expression pattern from what was observed in wild type mice.

The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent pharmacological and genetic evidence suggests that melanocortin receptor (MCR) signaling modulates neurobiological responses to ethanol and ethanol intake. Agouti-related protein (AgRP) is synthesized by neurons in the arcuate nucleus of the hypothalamus and is a natural antagonist of MCRs. Because central administration of the functionally active AgRP fragment AgRP-(83-132) increases ethanol intake by C57BL/6 J mice, we determined if mutant mice lacking normal production of AgRP (AgRP(-/-)) and maintained on a C57BL/6 J genetic background would show reduced self-administration of ethanol relative to littermate wild-type (AgRP(+/+)) mice. AgRP(-/-) mice showed reduced 8% (v/v) ethanol-reinforced lever-pressing behavior relative to AgRP(+/+) mice in daily 2-h sessions, but normal sucrose-, saccharin- and water-reinforced lever-pressing. Similarly, AgRP(-/-) mice showed reduced consumption of 8% ethanol in a two-bottle limited access test (2 h/day), although this effect was largely sex-dependent. Using drinking-in-the-dark (DID) procedures, AgRP(-/-) mice showed blunted binge-like drinking of 20% (v/v) ethanol which was associated with lower blood ethanol levels (85 mg/dl) relative to AgRP(+/+) mice (133 mg/dl) after 4 h of intake. AgRP(-/-) mice showed normal ethanol metabolism and did not show altered sensitivity to the sedative effects of ethanol. These observations with genetically altered mice are consistent with previous pharmacological data and suggest that endogenous AgRP signaling modulates the reinforcing properties of ethanol and binge-like ethanol drinking.

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins, and they are considered major targets of approximately half of all therapeutic agents. Human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and its mutants are directly related to severe human obesity. Here, we describe optimized protocols for the high-yield expression and purification of hMC4R that will accelerate structural study. Truncations of the N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion increase the solubility as well as stability of the protein. Strikingly, co-expression of human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells enables excellent stability of hMC4R. The protein yield in the human mini-AgRP co-expression system is increased by about 3-4 times compared to that of hMC4R alone. Data from analytical size exclusion chromatography (aSEC) and thermostability assay show that hMC4R becomes homogeneous and stable with a melting temperature of 58°C in the presence of human mini-AgRP.

Melanocortin-1 receptor (MC1R) and its ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan-a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio, and a tan-yellow color in pelletted cells. Moreover, ASIP-treated cells showed reduced proliferation and a melanoblast-like appearance, seen also in melanocyte lines from yellow (A(y)/a and Mc1r(e)/ Mc1r(e)) mice. However ASIP-YY, a C-terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP-YY inhibited the cAMP rise induced by alphaMSH analog NDP-MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn(mg-3J/mg-3J) or Mgrn1(md-nc/md-nc)) also responded to both ASIP and ASIP-YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP-MC1R signaling includes a cAMP-independent pathway through attractin and mahogunin, while the known cAMP-dependent component requires neither attractin nor mahogunin.

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

Circadian phase shifts in peripheral clocks induced by changes in feeding rhythm often result in insulin resistance. However, whether the hypothalamic control system for energy metabolism is involved in the feeding rhythm-related development of insulin resistance is unknown. Here, we show the physiological significance and mechanism of the involvement of the agouti-related protein (AgRP) in evening feeding-associated alterations in insulin sensitivity. Evening feeding during the active dark period increased hypothalamic AgRP expression and skeletal muscle insulin resistance in mice. Inhibiting AgRP expression by administering an antisense oligo or a glucocorticoid receptor antagonist mitigated these effects. AgRP-producing neuron-specific glucocorticoid receptor-knockout (AgRP-GR-KO) mice had normal skeletal muscle insulin sensitivity even under evening feeding schedules. Hepatic vagotomy enhanced AgRP expression in the hypothalamus even during ad-lib feeding in wild-type mice but not in AgRP-GR-KO mice. The findings of this study indicate that feeding in the late active period may affect hypothalamic AgRP expression via glucocorticoids and induce skeletal muscle insulin resistance.

Agouti-related peptide (AgRP) neurons of the hypothalamus play a role in hunger-triggered food intake, stability of body weight, and long-term energy balance. A recent study showed that activation of the Gs-linked G protein-coupled receptors (GCPR) expressed by hypothalamic AgRP neurons promotes a sustained increase in food intake. Enhanced AgRP release has been the postulated underlying mechanism. Here, we confirmed that activation of Gs-coupled receptors expressed by AgRP neurons in the arcuate nucleus (ARC) of the hypothalamus, which is the primary brain region for the synthesis and release of AgRP, leads to increased release of AgRP in the paraventricular nucleus of the hypothalamus (PVN). We were unable to confirm changes in AgRP expression or intracellular content using traditional histological techniques. Thus, we developed an assay to measure AgRP in the extracellular fluid in the brain using large molecular weight cut-off microdialysis probes. Our technique enables assessment of brain AgRP pharmacokinetics under physiological conditions and in response to specific pharmacological interventions designed to modulate AgRP signaling.

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida's Gulf and Atlantic coasts as camouflage from predators. To facilitate genomic analyses, we first generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus. Next, in a uniquely variable mainland population (Peromyscus polionotus albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that pigment variation is strongly associated with an ∼2-kb region ∼5 kb upstream of the Agouti signaling protein coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000 km. Together, our results suggest that this identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.

The study was undertaken considering age-related changes in susceptibility to experimental autoimmune encephalomyelitis (EAE) and a putative role of spleen in pathogenesis of this disease. The phenotypic and functional characteristics of T splenocytes were examined in young (3-month-old), middle-aged (8-month-old) and aged (26-month-old) Dark Agouti rats immunized for EAE with rat spinal cord in complete Freund's adjuvant. The rat susceptibility to EAE induction, as well as the number of activated CD4+CD134+ lymphocytes retrieved from their spinal cords progressively decreased with aging. To the contrary, in rats immunized for EAE the number of activated CD4+ splenocytes, i.e., CD4+CD134+, CD4+CD25+FoxP3- and CD4+CD40L+ cells, progressively increased with aging. This was associated with age-related increase in (i) CD4+ splenocyte surface expression of CD44, the molecule suggested to be involved in limiting emigration of encephalitogenic CD4+ cells from spleen into blood and (ii) frequency of regulatory T cells, including CD4+CD25+FoxP3+ cells, which are also shown to control encephalitogenic cell migration from spleen into the central nervous system. In favor of expansion of T-regulatory cell pool in aged rats was the greater concentration of IL-10 in unstimulated, Concanavalin A (ConA)- and myelin basic protein (MBP)-stimulated splenocyte cultures from aged rats compared with the corresponding cultures from young ones. Consistent with the age-related increase in the expression of CD44, which is shown to favor Th1 effector cell survival by interfering with CD95-mediated signaling, the frequency of apoptotic cells among CD4+ splenocytes, despite the greater frequency of CD95+ cells, was diminished in splenocyte cultures from aged compared with young rats. In addition, in control, as well as in ConA- and MBP-stimulated splenocyte cultures from aged rats, despite of impaired CD4+ cell proliferation, IFN-γ concentrations were greater than in corresponding cultures from young rats. This most likely reflected increased abundance of IFN-γ-producing cells in splenocyte cultures from aged compared with young rats. The diminished CD4+ cell proliferation in response to ConA and MBP in splenocyte cultures from aged compared with young rats could be, at least partly, associated with an enhanced splenic expression of iNOS mRNA in aged rats. Thus, the study suggests that age-associated changes leading to entrapping of activated CD4+ cells in the spleen could contribute to the restriction in development of EAE in aged rats.

The agouti gene encodes the agouti signaling protein (ASIP) which regulates pheomelanin and eumelanin synthesis in mammals. To investigate the role of agouti in coat color variation of alpaca, we characterized the agouti gene and identified three mutations potentially involved with the determinism of eumelanic and pheomelanic phenotypes. The exon-4 hosts the mutations g.3836C>T, g.3896G>A and g.3866_3923del57. Further analysis of these mutations revealed two genotypes for black animals. The reverse transcription analysis of mRNA purified from skin biopsies of alpaca revealed the presence of three transcripts with different 5' untranslated regions (UTRs) and color specific expression. The white specific transcript, possibly originating from a duplication event (intra-chromosomal recombination) of the agouti gene is characterise by a 5'UTR containing 142bp of the NCOA6 gene sequence. Furthermore, the relative level expression analysis of mRNA demonstrates that the agouti gene has up-regulated expression in white skin, suggesting a pleiotropic effect of agouti in the white phenotype. Our findings refine the structure of the agouti locus and transcripts and provide additional information in order to understand the role of agouti in the pigmentation of alpaca.

Cancer patients may experience symptom clusters, including chemotherapy-induced (CI) gut toxicity (CIGT) and cognitive impairment. Analgesic selection for pain associated with CIGT is difficult as opioids induce glial reactivity and unwanted side effects. This study quantified central glial reactivity and proinflammatory effects in rats with CIGT using three mechanistically different analgesics. Regional adaptations were indicative of immune-to-brain signaling routes. Utilizing a 5-fluorouracil-induced GT (5IGT) rat model and analgesic intervention (carprofen (CAR), buprenorphine (BUP), and tramadol (TRAM)), spinal and brain neuroimmune modulation was examined via microglial, astrocyte, and proinflammatory (cluster of differentiation molecule 11b; CD11b, glial fibrillary associated protein; GFAP, and interleukin-1 beta; IL1β) reactivity marker expression changes by western blot analysis. 5IGT significantly increased thoracic GFAP (p < 0.05) and IL-1β (p < 0.0001) expression, CAR and BUP ameliorated these effects. BUP and TRAM with 5-FU synergistically increased hippocampal GFAP expression. CAR administered with 5IGT significantly elevated hippocampal and thoracic CD11b expression levels (p < 0.05). The neuroimmune responses observed in this study suggest activation of peripheral-to-central immune signaling pathways. We speculate that the opioid-induced hippocampal changes inferred a humorally mediated mechanism, whereas thoracic neuroimmune modifications indicated activation of an indirect neural route. Although TRAM ameliorated 5IGT-intestinal inflammation, this opioid presents complications relating to bodyweight and regional glial dysregulation (neuroinflammation) and may not be optimal in the management of pain associated with 5IGT. The chemotherapy-induced gut-derived neuroimmune consequences observed suggest a potential mechanistic contribution to central components of the cancer symptom cluster experience, while the opioid-related glial changes have implications for optimal pain management in this setting warranting further investigation.

The melanocortin system is one of the most complex hormonal systems in vertebrates. Atypically, the signaling of melanocortin receptors is regulated by the binding of endogenous antagonists, named agouti-signaling protein (ASIP) and agouti-related protein (AGRP). Teleost specific genome duplication (TSGD) rendered new gene copies in teleost fish and up to four different genes of the agouti family of peptides have been characterized. In this paper, molecular cloning was used to characterize mRNA of the agouti family of peptides in sea bass. Four different genes were identified: AGRP1, ASIP1, AGRP2 and ASIP2. The AGRP1 gene is mainly expressed in the brain whereas ASIP1 is mainly expressed in the ventral skin. Both ASIP2 and AGRP2 are expressed in the brain and the pineal gland but also in some peripheral tissues. Immunocytochemical studies demonstrated that AGRP1 is exclusively expressed within the lateral tuberal nucleus, the homologue of the mammalian arcuate nucleus in fish. Long-term fasting (8-29 days) increased the hypothalamic expression of AGRP1 but depressed AGRP2 expression (15-29 days). In contrast, the hypothalamic expression of ASIP2 was upregulated during short-term fasting suggesting that this peptide could be involved in the short term regulation of food intake in the sea bass.