A plethora of molecular and functional studies in tetrapods has led to the discovery of multiple taste 1 receptor (
) genes encoding G-protein coupled receptors (
) responsible for sweet (
+
) and ...umami (
+
) taste. In fish, the
gene family repertoires greatly expanded because of several
gene duplications, and recent studies have shown
functional divergence from canonical mammalian sweet taste perceptions, putatively as an adaptive mechanism to develop distinct feeding strategies in highly diverse aquatic habitats. We addressed this question in the carnivore fish gilthead seabream (
), a model species of aquaculture interest, and found that the
gene repertoire consists of eight members including
,
and six
-
gene duplicates, adding further evidence to the evolutionary complexity of fish
families. To analyze
taste functions, we first developed a stable gene reporter system based on Ca
-dependent calcineurin/
signaling to examine specifically in vitro the responses of a subset of
heterodimers to L-amino acids (L-AAs) and sweet ligands. We show that although differentially tuned in sensitivity and magnitude of responses,
/
,
/
and
/
may equally serve to transduce amino acid taste sensations. Furthermore, we present preliminary information on the potential involvement of the G
protein alpha subunits
and
in taste signal transduction.
In this study, we investigated the transcriptional spatio-temporal dynamics of the taste 1 receptor (T1R) gene family repertoire in seabream (Sparus aurata sa), during larval ontogeny and in adult ...tissues. In early larval development, saT1R expression arises heterochronously, i.e. the extraoral taste-related perception in the gastrointestinal tract (GIT) anticipates first exogenous feeding (at 9 days post hatching dph), followed by the buccal/intraoral perception from 14 dph onwards, supporting the hypothesis that the early onset of the molecular machinery underlying saT1R expression in the GIT is not induced by food but rather genetically hardwired. During adulthood, we characterized the expression patterns of saT1R within specific tissues (n = 4) distributed in oropharingeal, GIT and brain regions substantiating their functional versatility as chemosensory signaling players to a variety of biological functions beyond oral taste sensation. Further, we provided for the first time direct evidences in fish for mRNA co-expression of a subset of saT1R genes (mostly saT1R3, i.e. the common subunit of the heterodimeric T1R complexes for the detection of “sweet” and “umami” substances), with the selected gut peptides ghrelin (ghr), cholecystokinin (cck), hormone peptide yy (pyy) and proglucagon (pg). Each peptide defines the enteroendocrine cells (ECCs) identity, and establishes on morphological basis, a direct link for T1R chemosensing in the regulation of fish digestive processes. Finally, we analyzed the spatial gene expression patterns of 2 taste signaling components functionally homologous to the mammalian G(i)α subunit gustducin, namely saG(i)α1 and saG(i)α2, and demonstrated their co-localization with the saT1R3 in EECs, thus validating their direct involvement in taste-like transduction mechanisms of the fish GIT. In conclusion, data provide new insights in the evolutionary conservation of gut sensing in fish suggesting a conserved role for nutrient sensors modulating entero-endocrine secretion.
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Abstract The present study reports the complete coding sequences for two paralogues for leptin (sLepA1 and sLepA2) and leptin receptor (sLepR) in Atlantic salmon. The deduced 171-amino acid (aa) ...sequence of sLepA1 and 175 aa sequence for sLepA2 shows 71.6% identity to each other and clusters phylogenetically with teleost Lep type A, with 22.4% and 24.1% identity to human Lep. Both sLep proteins are predicted to consist of four helixes showing strong conservation of tertiary structure with other vertebrates. The highest mRNA levels for sLepA1 in fed fish (satiation ration = 100%) were observed in the brain, white muscle, liver, and ovaries. In most tissues sLepA2 generally had a lower expression than sLepA1 except for the gastrointestinal tract (stomach and mid-gut) and kidney. Only one leptin receptor ortholog was identified and it shares 24.2% aa sequence similarity with human LepR, with stretches of highest sequence similarity corresponding to domains considered important for LepR signaling. The sLepR was abundantly expressed in the ovary, and was also high in the brain, pituitary, eye, gill, skin, visceral adipose tissue, belly flap, red muscle, kidney, and testis. Fish reared on a rationed feeding regime (60% of satiation) for 10 months grew less than control (100%) and tended to have a lower sLepA1 mRNA expression in the fat-depositing tissues visceral adipose tissue ( p < 0.05) and white muscle (n.s.). sLepA2 mRNA levels was very low in these tissues and feeding regime tended to affect its expression in an opposite manner. Expression in liver differed from that of the other tissues with a higher sLepA2 mRNA in the feed-rationed group ( p < 0.01). Plasma levels of sLep did not differ between fish fed restricted and full feeding regimes. No difference in brain sLepR mRNA levels was observed between fish fed reduced and full feeding regimes. This study in part supports that sLepA1 is involved in signaling the energy status in fat-depositing tissues in line with the mammalian model, whereas sLepA2 may possibly play important roles in the digestive tract and liver. At present, data on Lep in teleosts are too scarce to allow generalization about how the Lep system is influenced by tissue-specific energy status and, in turn, may regulate functions related to feed intake, growth, and adiposity in fish. In tetraploid species like Atlantic salmon, different Lep paralogues seems to serve different physiological roles.
Similar to many marine teleost species, Atlantic cod undergo remarkable physiological changes during the early life stages with concurrent and profound changes in feeding biology and ecology. In ...contrast to the digestive system, very little is known about the ontogeny and the localization of the centers that control appetite and feed ingestion in the developing brain of fish. We examined the expression patterns of three appetite regulating factors (orexigenic: neuropeptide Y, NPY; prepro-orexin, pOX and anorexigenic: cocaine- and amphetamine-regulated transcript, CART) in discrete brain regions of developing Atlantic cod using chromogenic and double fluorescent in situ hybridization. Differential temporal and spatial expression patterns for each appetite regulator were found from first feeding (4 days post hatch; dph) to juvenile stage (76 dph). Neurons expressing NPY mRNA were detected in the telencephalon (highest expression), diencephalon, and optic tectum from 4 dph onward. CART mRNA expression had a wider distribution along the anterior-posterior brain axis, including both telencephalon and diencephalon from 4 dph. From 46 dph, CART transcripts were also detected in the olfactory bulb, region of the nucleus of medial longitudinal fascicle, optic tectum and midbrain tegmentum. At 4 and 20 dph, pOX mRNA expression was exclusively found in the preoptic region, but extended to the hypothalamus at 46 and 76 dph. Co-expression of both CART and pOX genes were also observed in several hypothalamic neurons throughout larval development. Our results show that both orexigenic and anorexigenic factors are present in the telencephalon, diencephalon and mesencephalon in cod larvae. The telencephalon mostly contains key factors of hunger control (NPY), while the diencephalon, and particularly the hypothalamus may have a more complex role in modulating the multifunctional control of appetite in this species. As the larvae develop, the overall progression in temporal and spatial complexity of NPY, CART and pOX mRNAs expression might be correlated to the maturation of appetite control regulation. These observations suggest that teleost larvae continue to develop the regulatory networks underlying appetite control after onset of exogenous feeding.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic ...fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to elucidate the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity. In addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic analysis reveal that amino acid, monocarboxylates, ionic and vitamin transmembrane transporters were substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting augmented intestinal laxity that may enhance paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membraned tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.
Hox cluster organization represents a valuable marker to study the effects of recent genome duplication in salmonid fish (25-100 Mya). Using polymerase chain reaction amplification of cDNAs, BAC ...library screening, and genome walking, we reconstructed 13 Hox clusters in the Atlantic salmon containing 118 Hox genes including 8 pseudogenes. Hox paralogs resulting from the genome duplication preceding the radiation of ray-finned fish have been much better preserved in salmon than in other model teleosts. The last genome duplication in the salmon lineage has been followed by the loss of 1 of the 4 HoxA clusters. Four rounds of genome duplication after the vertebrate ancestor salmon Hox clusters display the main organizational features of vertebrate Hox clusters, with Hox genes exclusively that are densely packed in the same orientation. Recently, duplicated Hox clusters have engaged a process of divergence, with several cases of pseudogenization or asymmetrical evolution of Hox gene duplicates, and a marked erosion of identity in noncoding sequences. Strikingly, the level of divergence attained strongly depends on the Hox cluster pairs rather than on the Hox genes within each cluster. It is particularly high between both HoxBb clusters and both HoxDa clusters, whereas both HoxBa clusters remained virtually identical. Positive selection on the Hox protein-coding sequences could not be detected.
We examined the distribution of Neuropeptide Y (NPY), cocaine‐ and amphetamine‐regulated transcript (CART) and prepro‐orexin (pOX) in the brain of Atlantic cod by in situ hybridization from onset of ...exogenous feeding (4days post hatch, dph) to postlarval stage (76dph). All examined genes were expressed from 4dph and displayed temporal and spatial differences in expression during development. pOX mRNA was only expressed in hypothalamus. NPY and CART mRNA expressions had broad distributions in the brain, including telencephalon, diencephalon, and optic tectum. In addition, NPY transcripts were found in the preoptic region, while CART was found in medulla oblongata. New populations of CART positive neurons appeared in olfactory bulb, in the region of the nucleus of medial longitudinal fascicle, and later in midbrain tegmentum. We found several examples of co‐expression (NPY‐CART) and (CART‐pOX). Distributions of NPY and pOX transcripts suggest that telencephalon and hypothalamus are involved in stimulation of feeding in cod. Expression of CART indicates that “satiety centers” are located in hypothalamus, thalamus and medulla oblongata.
Grant Funding Source: Supported by Research Council of Norway‐ CODE 199482
Highlights ► Cloning and identification of the prop1 homeobox gene in zebrafish and salmon. ► Demonstration of pituitary-specific expression of prop1 in both species. ► Induced knock down by ...administration of morpholino oligonucleotides in zebrafish. ► Analysis of knock down effects on known target genes (in mammals) and others expressed during fish pituitary development, by in situ hybridization, qPCR and morphology. ► Conservation of prop1 mediated pit1 pathway is extended to fish; discovery of additional prop1 targets (lim3 homeobox gene).
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