Raúl Cortés

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.

Characterization, tissue distribution and regulation by fasting of the agouti family of peptides in the sea bass (Dicentrarchus labrax)

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.

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.

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 β-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.

Involvement of melanocortin receptor accessory proteins (MRAPs) in the function of melanocortin receptors.

The melanocortin system integrates different agonists, competitive or inverse agonists, and receptors. Recent investigations have also discovered a specific system of melanocortin receptor accessory proteins (MRAPs) that are involved in the regulation of the functional expression of these receptors. MRAP1 mutations are responsible for type 2 familial glucocorticoid deficiency (FGD2), a rare autosomal disorder characterized by high plasma adrenocorticotropin hormone (ACTH) levels but severe cortisol deficiency. ACTH binds melanocortin 2 receptor (MC2R), a G protein-coupled receptor, in the adrenal gland to promote corticosteroid synthesis. In the absence of MRAP1, MC2R cannot translocate from the endoplasmic reticulum to the plasma membrane and ACTH-induced signaling is extinguished. A second MRAP protein, called MRAP2, also modulates MC2R activity. MRAPs also interact with the other melanocortin receptors, adjusting their pharmacological properties. In this paper, we briefly review the MRAP system and its interaction with melanocortin receptors.

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.

Effects of acute handling stress on short-term central expression of orexigenic/anorexigenic genes in zebrafish

Physiological mechanisms driving stress response in vertebrates are evolutionarily conserved. These mechanisms involve the activation of both the hypothalamic-sympathetic-chromaffin cell (HSC) and the hypothalamic-pituitary-adrenal (HPA) axes. In fish, the reduction of food intake levels is a common feature of the behavioral response to stress but the central mechanisms coordinating the energetic response are not well understood yet. In this work, we explore the effects of acute stress on key central systems regulating food intake in fish as well as on total body cortisol and glucose levels. We show that acute stress induced a rapid increase in total body cortisol with no changes in body glucose, at the same time promoting a prompt central response by activating neuronal pathways. All three orexigenic peptides examined, i.e., neuropeptide y (npy), agouti-related protein (agrp), and ghrelin, increased their central expression level suggesting that these neuronal systems are not involved in the short-term feeding inhibitory effects of acute stress. By contrast, the anorexigenic precursors tested, i.e., cart peptides and pomc, exhibited increased expression after acute stress, suggesting their involvement in the anorexigenic effects.

In vitro effect of Levamisole on Rainbow Trout (Oncorhynchus mykiss) peripheral blood mononuclear cells

Se estudio el efecto in vitro del levamisol (1, 10 y 100 μg/ml) sobre celulas de la respuesta inmune innata de trucha arcoiris (Oncorhynchus mykiss), particularmente la actividad de las celulas citotoxicas naturales y de los macrofagos presentes en sangre periferica. El efecto del levamisol sobre las celulas citotoxicas naturales fue evaluado mediante su capacidad para lisar celulas tumorales HL-60, mientras que la actividad de los macrofagos fue evaluado mediante la produccion de especies reactivas del oxigeno y de oxido nitrico. Los resultados indican que la actividad de las celulas citotoxicas naturales fue incrementada significativamente por 1 μg/ml levamisol mientras que dosis mayores no produjeron este efecto. Por otro lado, la produccion de especies reactivas del oxigeno y de oxido nitrico por los macrofagos no fue incrementada por ninguna de las dosis usadas, sin embargo dosis de 100 μg/ml indujeron una disminucion significativa en los niveles de oxido nitrico, lo cual podria ser deletereo para la respuesta defensiva de estos peces.

Effect of PHA and LPS on the Activity of Nonspecific Cytotoxic Cells and Macrophages of Rainbow Trout (Oncorhynchus mykiss)

En este trabajo evaluamos el efecto de la lectina fitohemaglutinina (PHA) y del lipopolisacarido bacteriano (LPS) sobre la actividad de las celulas citotoxicas naturales (NCCs) y de los macrofagos de la sangre periferica de la truchas arcoiris. Las NCCs son celulas de la inmunidad innata, homologas a las celulas Natural Killer (NK) de mamiferos, capaces de eliminar celulas infectadas con patogenos intracelulares y tambien celulas tumorales. Su actividad litica es mediada por las perforinas/granzymas y el sistema Fas receptor-Fas Ligando (FasR-FasL). La actividad de estas celulas fue evaluada por su capacidad para lisar celulas tumorales HL-60. Por otro lado, los macrofagos eliminan microrganismos mediante diversos mecanismos, los que incluyen la produccion de especies reactivas del oxigeno (ROS) y del oxido nitrico (NO). Nuestros resultados muestran que el PHA incrementa significativamente la actividad de las NCCs contra las celulas HL-60, aun bajo condiciones en las que el sistema litico de las perforinas/granzimas estaria suprimido, apoyan la existencia del sistema FasR-FasL en truchas. El PHA no mostro efecto alguno en la produccion de ROS y NO por los macrofagos. Finalmente, al evaluar el efecto del LPS en ambas poblaciones celulares, no se obtuvo efecto alguno tanto en la actividad citotoxica de las NCCs, como tampoco en la produccion de ROS y NO por los macrofagos.