Elisa Sánchez

Expression of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis genes during zebrafish Danio rerio early embryogenesis

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes, many of which are particularly vital during embryonic development. This study investigated the expression of genes encoding enzymes involved in LC-PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, during early embryonic development of zebrafish. First, zebrafish elovl2 cDNA was isolated and functionally characterised in yeast, showing high specificity towards C20- and C22-PUFAs, compared to C18 substrates. Second, spatial-temporal expression for elovl2 and the previously cloned fad and elovl5 were studied during zebrafish early embryonic development. Temporal expression shows that all three genes are expressed from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. However, a complete activation of the biosynthetic pathway seems to be delayed until 12 hpf, when noticeable increases of fad and elovl2 transcripts were observed, in parallel with high docosahexaenoic acid levels in the embryo. Spatial expression was studied by whole-mount in situ hybridisation in 24 hpf embryos, showing that fad and elovl2 are highly expressed in the head area where neuronal tissues are developing. Interestingly, elovl5 shows specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues analysed, with liver, intestine and brain showing the highest expression.

Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin–CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

Role of melanocortin receptor accessory proteins in the function of zebrafish melanocortin receptor type 2

In this paper, we identify three different MRAPs in zebrafish, zfMRAP1, zfMRAP2a and zfMRAP2b, and demonstrate that zfMC2R is not functional in the absence of MRAP expression. ZfMRAP1 expression was restricted to adipose tissue and the anterior kidney whereas MRAP2a and MRAP2b were expressed in all the tissues tested. Quantification of surface receptor and immunofluorescence studies indicated that the receptor is unable to translocate to membrane in the absence of MRAP isoforms. MRAP1 and MRAP2b are localized in the plasma membrane in the absence of zfMC2R expression but MRAP2b is retained in perinuclear position. MRAP1 and MRAP2a displayed an equivalent translocation capacity to the membrane of zfMC2R but only zfMRAP1 expression led to intracellular cAMP increases after ACTH stimulation. ZfMRAP2b had no effect on zfMC2R activity but both zfMRAP2 isoforms enhanced the zfMRAP1-assisted cAMP intracellular increase, suggesting an interaction between zfMRAP1 and zfMRAP2s when regulating zfMC2R activity.

Fish melanocortin system

Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC(1)-MC(5)) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC(5) receptor, while pufferfish have 4 receptors with no melanocortin MC(3) receptor and one copy of melanocortin MC(5) receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC(2) receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC(1) receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC(1) receptor are responsible for reduced melanization. Both melanocortin MC(4) and MC(5) receptor are expressed in the hypothalamus, and central melanocortin MC(4) receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC(2) and MC(5) receptors. Both sea bass melanocortin MC(1) and MC(4) receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively.

Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development

The oocytes of many fish species accumulate high amounts of neutral lipids as a caloric reserve for embryonic and larval development. We propose that lipoprotein lipase (LPL, EC 3.1.1.34) plays an important role in supplying the oocytes with fatty acids and we have cloned its cDNA from the ovary of sea bass, and determined the patterns of LPL activity and LPL mRNA expression in the ovary. The cDNA obtained was 3051 bp long with an open reading frame encoding 518 amino acids. The amino acid sequence has a high similarity and shows similar structural features to LPL of other species. Northern blot analysis revealed LPL expression in adipose tissue and gonads only. LPL activity and LPL mRNA expression in the ovary was very high in fish with a gonadosomatic index (GSI) above 5, coinciding with the appearance of a high number of lipid droplets in the ooplasm. The LPL mRNA expression was localised to the follicle cells surrounding the oocyte. Our results suggest that LPL is likely to play an important role in the incorporation of neutral lipids into the oocytes, and that follicle cells, in addition to participating in steroidogenesis, also may be important in building up oocyte lipid reserves.

Phosphodiesterase inhibitor-dependent inverse agonism of agouti-related protein on melanocortin 4 receptor in sea bass ( Dicentrarchus labrax )

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor mainly expressed in the central nervous system of vertebrates. Activation of the MC4R leads to a decrease in food intake, whereas inactivating mutations are a genetic cause of obesity. The binding of agouti-related protein (AGRP) reduces not only agonist-stimulated cAMP production (competitive antagonist) but also the basal activity of the receptor, as an inverse agonist. Transgenic zebrafish overexpressing AGRP display increased food intake and linear growth, indicative of a physiological role for the melanocortin system in the control of the energy balance in fish. We report on the cloning, pharmacological characterization, tissue distribution, and detailed brain mapping of a sea bass (Dicentrarchus labrax) MC4R ortholog. Sea bass MC4R is profusely expressed within food intake-controlling pathways of the fish brain. However, the activity of the melanocortin system during progressive fasting does not depend on the hypothalamic/pituitary proopiomelanocortin (POMC) and MC4R expression, which suggests that sea bass MC4R is constitutively activated and regulated by AGRP binding. We demonstrate that AGRP acts as competitive antagonist and reduces MTII-induced cAMP production. AGRP also decreases the basal activity of the receptor as an inverse agonist. This observation suggests that MC4R is constitutively active and supports the evolutionary conservation of the AGRP/MC4R interactions. The inverse agonism, but not the competitive antagonism, depends on the presence of a phosphodiesterase inhibitor (IBMX). This suggests that inverse agonism and competitive antagonism operate through different intracellular signaling pathways, a view that opens up new targets for the treatment of melanocortin-induced metabolic syndrome.

Melatonin-synthesizing enzymes in pineal, retina, liver, and gut of the goldfish (Carassius): mRNA expression pattern and regulation of daily rhythms by lighting conditions

It has been suggested that melatonin is synthesized in nonphotosensitive organs of vertebrates in addition to the well-known sites of the pineal gland and retina. However, very few studies have demonstrated the gene expression of melatonin-synthesizing enzymes in extrapineal and extraretinal locations. This study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), in central and peripheral locations of a teleost fish, the goldfish (Carassius auratus). First, the full-length cDNA sequences corresponding to the goldfish AANAT-2 (gAanat-2) and HIOMT-2 (gHiomt-2) were cloned, showing high similarity with other teleost sequences. Two forms of AANAT exist in teleosts. Here, for the first time, two isoforms of HIOMT are deduced from phylogenetic analysis. Moreover, both HIOMT and AANAT were detected in several peripheral locations, including liver and gut, the present results being the first to find HIOMT in nonphotosensitive structures of a fish species. Second, quantitative real-time polymerase chain reaction (PCR) studies were performed to investigate regulation of gAanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions. The current results show circadian rhythms in Aanat-2 and Hiomt-2 transcripts in liver and hindgut, suggesting a local melatonin synthesis in goldfish. Moreover, the analysis of daily expression of gAanat-2 under different lighting conditions, including continuous light (24L) and darkness (24D) revealed light-dependent rhythms in the pineal and retina, as expected, but also in liver and hindgut. The persistence in hindgut of these gAanat-2 rhythms under both constant conditions, 24L and 24D, suggests expression of this transcript is governed by a circadian clock and entrained by nonphotic cues. Finally, the current results support the existence of melatonin synthesis in gut and liver of the goldfish. (Author correspondence: mjdelgad@bio.ucm.es)

Characterization of the sea bass melanocortin 5 receptor: a putative role in hepatic lipid metabolism

SUMMARY The melanocortin 5 receptor (MC5R) plays a key role in the regulation of exocrine secretion in mammalian species. This receptor has also been characterized in some fish species but its function is unknown. We report the molecular and pharmacological characterization, as well as the tissue expression pattern, of sea bass MC5R. Cloning of five active alleles showing different levels of sensitivity to endogenous melanocortin and one non-functional allele demonstrate the allelic complexity of the MC5R locus. The sea bass receptor was activated by all the melanocortins tested, with ACTH and desacetyl-MSH and β-MSH showing the lowest efficiency. The acetylation of the MSH isoforms seems to be critical for the effectiveness of the agonist. Agouti-related protein had no effect on basal or agonist-stimulated activation of the receptor. SbMC5R was mainly expressed in the brain but lower expression levels were found in several peripheral tissues, including liver. Progressive fasting did not induce up- or downregulation of hypothalamic MC5R expression, suggesting that central MC5R is not involved in the regulation of food intake in the sea bass. MTII, a sbMC5R agonist, stimulated hepatic lipolysis in vitro, measured as free fatty acid release into the culture medium after melanocortin agonist exposure of liver fragments, suggesting that MC5R is involved in the regulation of hepatic lipid metabolism. Taken together, the data suggest that different allelic combinations may confer differential sensitivity to endogenous melanocortin in tissues where MC5R is expressed and, by extension, in hepatic lipid metabolism.

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 and pharmacological characterization of the melanocortin type 1 receptor in the sea bass

Melanocortin 1 receptor (MC1R) plays a key role in the physiology of the vertebrate pigment system. Point mutations producing hyperactive or inactive receptors result in darkening or paling effects, respectively. We report the molecular and pharmacological characterization, as well as the tissue expression pattern, of the sea bass Mc1r. Similar to other MC1Rs, the sea bass gene is highly polymorphic and nine DNA polymorphisms, seven of them involving an amino acid substitution, were detected. SbMc1r is mainly expressed in the testis, fat and liver with moderate levels in the ventral and dorsal skin. The sea bass receptor was activated by all the melanocortins tested, with ACTH showing the lowest efficiency. The acetylation level of the MSH isoforms seems to be critical for the effectively of the agonist. Agouti-related protein (AGRP) drastically inhibited the basal activity of the receptor in vitro, as an inverse agonist does, but only in the presence of phosphodiesterase inhibitors. This observation suggests that sbMc1r is constitutively activated and inversely regulated by AGRP, which is expressed in the skin of different fish species.