Behind melanocortin antagonist overexpression in the zebrafish brain: A behavioral and transcriptomic approach.

Melanocortin signaling is regulated by the binding of naturally occurring antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) that compete with melanocortin peptides by binding to melanocortin receptors to regulate energy balance and growth. Using a transgenic model overexpressing ASIP, we studied the involvement of melanocortin system in the feeding behaviour, growth and stress response of zebrafish. Our data demonstrate that ASIP overexpression results in enhanced growth but not obesity. The differential growth is explained by increased food intake and feeding efficiency mediated by a differential sensitivity of the satiety system that seems to involve the cocaine- and amphetamine- related transcript (CART). Stress response was similar in both genotypes. Brain transcriptome of transgenic (ASIP) vs wild type (WT) fish was compared using microarrays. WT females and males exhibited 255 genes differentially expressed (DEG) but this difference was reduced to 31 after ASIP overexpression. Statistical analysis revealed 1122 DEG when considering only fish genotype but 1066 and 981 DEG when comparing ASIP males or females with their WT counterparts, respectively. Interaction between genotype and sex significantly affected the expression of 97 genes. Several neuronal systems involved in the control of food intake were identified which displayed a differential expression according to the genotype of the fish that unravelling the flow of melanocortinergic information through the central pathways that controls the energy balance. The information provided herein will help to elucidate new central systems involved in control of obesity and should be of invaluable use for sustaining fish production systems.

Melanocortin receptor accessory protein 2 (MRAP2) interplays with the zebrafish melanocortin 1 receptor (MC1R) but has no effect on its pharmacological profile.

The melanocortin system is probably one of the most complex hormonal systems since it integrates agonist, encoded in the proopiomelanocortin precursor, endogenous antagonist, agouti signaling protein and agouti-related protein, five different G-protein coupled receptors and two accessory proteins. These accessory proteins interact with melanocortin receptors to allow traffic to the plasma membrane or to regulate the pharmacological profile. The MC1R fill the extension locus, which is primarily responsible for the regulation of pigmentation. In zebrafish, both MC1R and MRAP2 system are expressed in the skin. We demonstrate that zebrafish MC1R physically, or closely, interacts with the MRAP2 system, although this interaction did not result in modification of the studied pharmacological profile. However, progressive fasting induced skin darkening but also an upregulation of the MRAP2 expression in the skin, suggesting an unknown role for MRAP2a that could involve receptor desensitization processes. We also demonstrate that crowding stress induces skin darkening and a downregulation of MC1R expression in the skin.

Evolution of the melanocortin system.

The melanocortin system is one of the most complex of the hormonal systems. It involves different agonists encoded in the multiplex precursor proopiomelanocortin (POMC) or in different genes as ß-defensins, endogenous antagonist, like agouti-signalling protein (ASIP) or agouti-related protein (AGRP), and five different melanocortin receptors (MCRs). Rounds of whole genome duplication events have preceded the functional and molecular diversification of the family in addition some co-evolutionary and tandem duplication processes have been proposed. The evolutionary patterns of the different partners are controversial and different hypotheses have emerged from a study of the sequenced genomes. In this review, we summarize the different evolutionary hypotheses proposed for the different melanocortin partners.

Growth Performance After Agouti-Signaling Protein 1 (Asip1) Overexpression in Transgenic Zebrafish.

The melanocortin system is a key structure in the regulation of energy balance. Overexpression of inverse agonists, agouti-signaling protein (ASIP), and agouti-related protein (AGRP) results in increased food intake, linear growth, and body weight. ASIP regulates dorsal-ventral pigment polarity through melanocortin 1 receptor (MC1R) and overexpression induces obesity in mice by binding to central MC4R. Asip1 overexpression in transgenic zebrafish (asip1-Tg) enhances growth, yet experiments show fish overexpressing Asip1 do not develop obesity even under severe feeding regimes. Asip1-Tg fish do not need to eat more to grow larger and faster; thus, increased food efficiency can be observed. In addition, asip1-Tg fish reared at high density are able to grow far more than wild-type (WT) fish reared at low density, although asip1-Tg fish seem to be more sensitive to crowding stress than WT fish, thus making the melanocortin system a target for sustainable aquaculture, especially as the U.S. Food and Drug Association has recently approved transgenic fish trading.

Thyroid Hormones Regulate Zebrafish Melanogenesis in a Gender-Specific Manner.

Zebrafish embryos are treated with anti-thyroidal compounds, such as phenylthiourea, to inhibit melanogenesis. However, the mechanism whereby the thyroidal system controls melanin synthesis has not been assessed in detail. In this work, we tested the effect of the administration of diets supplemented with T3 (500µg/g food) on the pigment pattern of adult zebrafish. Oral T3 induced a pronounced skin paling in both adult female and male zebrafish that was reversible upon cessation of treatment. The number of visible melanophores was significantly reduced in treated fish. Accordingly, treatment down-regulated expression of tyrosinase-related protein 1 in both sexes. We also found sexually dimorphic regulation of some melanogenic genes, such as Dct/Tyrp2 that was dramatically up-regulated in females after T3 treatment. Thus, we demonstrated that melanogenesis is reversibly inhibited by thyroid hormones in adult zebrafish and make the discovery of gender-specific differences in the response of melanogenic gene expression. Thus, fish gender is now shown to be an important variable that should be controlled in future studies of fish melanogenesis.

Pigment patterns in adult fish result from superimposition of two largely independent pigmentation mechanisms.

Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern.

Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2.

Melanocortin 2 receptor (MC2R) is the only canonical ACTH receptor. Its functional expression requires the presence of an accessory protein, known as melanocortin receptor 2 accessory protein 1 (MRAP1). The vertebrate genome exhibits a paralogue gene called MRAP2, which is duplicated in zebrafish (MRAP2a and MRAP2b), although its function remains unknown. In this paper, we demonstrate that MRAP2a enables MC4R, a canonical MSH receptor, to be activated by ACTH with a similar sensitivity to that exhibited by MC2R. Both proteins physically interact and are coexpressed in the neurons of the preoptic area, a key region in the control of the energy balance and hypophyseal secretion in fish. ACTH injections inhibit food intake in wild-type zebrafish but not in fish lacking functional MC4R. Both MRAP1 and MRAP2a are hormonally regulated, suggesting that these proteins are substrates for feed-back regulatory pathways of melanocortin signaling. Fasting has no effect on the central expression of MRAP2a but stimulates MRAP2b expression. This protein interacts and is colocalized with MC4R in the tuberal hypothalamic neurons but has no effect on the pharmacologic profile of MC4R. However, MRPA2b is able to decrease basal reporter activity in cell lines expressing MC4R. It is plausible that MRAP2b decreases the constitutive activity of the MC4R during fasting periods, driving the animal toward a positive energy balance. Our data indicate that MRAP2s control the activity of MC4R, opening up new pathways for the regulation of melanocortin signaling and, by extension, for the regulation of the energy balance and obesity.

Molecular characterization and functional regulation of melanocortin 2 receptor (MC2R) in the sea bass. A putative role in the adaptation to stress.

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release.

Transient ectopic overexpression of agouti-signalling protein 1 (asip1) induces pigment anomalies in flatfish.

While flatfish in the wild exhibit a pronounced countershading of the dorso-ventral pigment pattern, malpigmentation is commonly observed in reared animals. In fish, the dorso-ventral pigment polarity is achieved because a melanization inhibition factor (MIF) inhibits melanoblast differentiation and encourages iridophore proliferation in the ventrum. A previous work of our group suggested that asip1 is the uncharacterized MIF concerned. In order to further support this hypothesis, we have characterized asip1 mRNAs in both turbot and sole and used deduced peptide alignments to analyze the evolutionary history of the agouti-family of peptides. The putative asip precursors have the characteristics of a secreted protein, displaying a putative hydrophobic signal. Processing of the potential signal peptide produces mature proteins that include an N-terminal region, a basic central domain with a high proportion of lysine residues as well as a proline-rich region that immediately precedes the C-terminal poly-cysteine domain. The expression of asip1 mRNA in the ventral area was significantly higher than in the dorsal region. Similarly, the expression of asip1 within the unpigmented patches in the dorsal skin of pseudoalbino fish was higher than in the pigmented dorsal regions but similar to those levels observed in the ventral skin. In addition, the injection/electroporation of asip1 capped mRNA in both species induced long term dorsal skin paling, suggesting the inhibition of the melanogenic pathways. The data suggest that fish asip1 is involved in the dorsal-ventral pigment patterning in adult fish, where it induces the regulatory asymmetry involved in precursor differentiation into mature chromatophore. Adult dorsal pseudoalbinism seems to be the consequence of the expression of normal developmental pathways in an inaccurate position that results in unbalanced asip1 production levels. This, in turn, generates a ventral-like differentiation environment in dorsal regions.

Stress-induced effects on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): a self-feeding approach.

Repetitive aquaculture-related protocols may act as cyclic stressors that induce chronic stress in cultured fish. The sea bass is particularly sensitive to stressful conditions and the mere presence of humans will disturb feeding behavior. In this paper, we study whether chronic stress induced by repetition of acute stress protocols affects long-term feeding behavior and growth performance in sea bass and whether exogenous cortisol may induce stress-like changes in these parameters. We demonstrate that both chronic stress and dietary cortisol decrease food intake and have a negative effect on feed conversion efficiency, severely impairing sea bass performance. Both experimental approaches induced changes in the daily feeding activity by lengthening the active feeding periods. Fish subjected to a cyclic stressor modify their daily feeding pattern in an attempt to avoid interference with the time of the stressor. The delay in feeding when fish are acutely and repeatedly stressed could be of substantial adaptive importance.